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Gary Ruvkun

The Ruvkun lab has explored two major themes: regulation by microRNA genes and other small RNAs, and control of longevity and metabolism by insulin and other endocrine pathways. Thousands of miRNAs have been discovered and are now implicated in control of gene expression of across eukaryotic phylogeny.
Saturation genetic analysis of the miRNA and RNAi pathways by the Ruvkun lab has revealed many of the protein cofactors that may mediate steps in how miRNAs and siRNAs engage their targets. These miRNA pathway protein cofactors could mediate steps in the recognition of the miRNA::mRNA RNA duplex to down-regulate translation. There is also increasing evidence of a cell biology of RNA regulation, so a more complex choreography is likely. The Ruvkun lab also discovered that there is complex negative regulation of RNAi and competing small RNA pathways. In addition to revealing fundamental regulatory axes in biology, some of these components may be developed as drug targets to enhance RNAi in mammals, a technical improvement that may be necessary to elevate a laboratory tool to a therapeutic modality.

The Ruvkun lab discovered many elements of the insulin-like signaling pathway controls C. elegans metabolism and longevity. Saturation genetic analysis of the pathway in the Ruvkun laboratory identified most of the signaling components, including daf-16, a Forkhead transcription factor that illuminated the function of the mammalian FoxO transcription factors, now intensively studied as insulin signaling transcriptional outputs. Recent insulin signaling mutant analyses in mouse and humans have validated the generality of these discoveries to other animals. These findings are also important for the eventual treatment of diabetes, a disease of insulin signaling. Dr. Ruvkun’s lab has also used full genome RNAi libraries to explore the comprehensive set of genes that regulate aging and metabolism. Many of these genes are broadly conserved in animal phylogeny and are likely to reveal the neuroendocrine system that assesses and regulates energy stores and assigns metabolic pathways based on that status. This study revealed other arrest points induced by gene inactivations that are as highly regulated and as key to lifespan regulation as the insulin regulated arrest point. Many of the gene inactivations that cause increased survival of arrested larvae encode the core conserved elements of cells that are targeted by antibiotics, which are produced by a wide range of fungi and microbes that animals encounter. Inactivation of these core genes may induce an endocrine state of drug detoxification and longevity induction. This xenobiotic regulatory axis has important and surprising implications for human health: the endocrine states that are normally induced by poisons and variation in the conserved detoxification response pathway may underlie diseases not normally thought of as xenobiotic response dysregulations.

Team

Maria Armakola
Research Fellow
Peter Breen
Research Technician
Christopher Carr
Research Scientist
Sylvia Fischer
Assistant in Molecular Biology
Amaranath (Jothi) Govindan
Research Associate
Elamparithi Jayamani
Research Fellow
Marina Kniazeva
Assistant in Molecular Biology
Nicolas Lehrbach
Research Fellow
Kai Mao
Research Fellow
Joshua Meisel
Research Fellow
Angel Morarro
Research Assistant
Martin Newman
Research Fellow
Alexandra Pontefract
Postdoctoral Associate
Kendall Saboda
Research Assistant
Diane Sacchetti
Administrative/Grant Coordinator
Jacapo Tani
Postdoctoral Associate
Kurt Warnhoff
Research Fellow
Wei Wei
Research Fellow
Prema Arasu
CEO and Vice Provost Kansas State University, Olathe
Kaveh Ashrafi
Associate Professor, Dept of Physiology, UCSF
Ralf Baumeister
University of Freiburg, Germany
Thomas Burglin
Associate Professor, Karolinska Institute, Stockholm
Maurice Butler
Associate Medical Director at BGB New York
Jacob Carlson
University of California, San Francisco
Annie Lee Conery
Clinical Trial Manager, MGH Translational Medicine Group
Sean Curran
Assistant Professor, University of Southern California
Nabanita De
Robert Dowen
Senior Research Associate, University of North Carolina
Joshua Elkington
University of California, Berkeley
Michael Finney
Managing Director, Finney Capital, San Francisco
Alison Frand
Assistant Professor, Biological Chemistry, UCLA
Harrison Gabel
Postdoctoral Fellow, Mike Greenberg Lab, Harvard Medical School
Susana Garcia
Institute of Biotechnology, University of Helsinki
Shoshanna Gottlieb
High school teacher, Philadelphia, PA
David Greenstein
Professor of Genetics, University of Minnesota
Ilho Ha
Research Director, Cancer Research Center, Theragen Etex, Suwon, Korea
Julie Hachey
Gabriel Hayes
Please see http://ccib.mgh.harvard.edu/ruvkun/Gabriel_Hayes_Remembrance
Oliver Hobert
Professor of Biochemistry and HHMI at Columbia University P&S
Patrick Hu
Assistant Professor, Life Sciences Institute, University of Michigan
Thomas Isenbarger
Attorney, Casimir Jones, Madison, WI
Scott Kennedy
Associate Professor, Dept. of Pharmacology, U Wisconsin
John Kim
Assistant Professor, Life Sciences Institute, University of Michigan
Koutarou Kimura
Assistant Professor, National Institute of Genetics, Osaka, Japan
Natasha Kirienko
Assistant Professor, Rice University
Allison Koweek
Personal organizer
Jonah Larkins-Ford
Graduate Student, Tufts University
Raymond Lee
Curator, C. elegans database, Caltech
Siu Sylvia Lee
Associate Professor, Dept. of Molecular Biology and Genetics, Cornell University
Weiqing Li
Research Scientist, University of Washington, now in Hangzhou, China
Clarissa Liu
Research Scientist, Theranos, Palo Alto
Ying Liu
Assistant Professor, Beijing University
Ho YI Mak
Assistant Investigator, Associate Professor, Hong Kong University of Science and Technology
Justine Melo
Visiting Assistant Professor, Haverford College
Taiowa Montgomery
Assistant Professor, Dept of Biology, Colorado State University
Jason Morris
Associate Professor, Fordham University, New York
Eyleen O'Rourke
Assistant Professor, Dept of Biology, University of Virginia
Scott Ogg
Vice President Development and Operations, Armo Biosciences, Redwood City, CA
Suzanne Paradis
Assistant Professor, Brandeis University
Devin Parry
Biology Teacher, The Lakeside School, Seattle
Amy Pasquinelli
Associate Professor, Dept. of Biology, UC San Diego
Garth Patterson
Assistant Dean, Rutgers University
Carolyn Phillips
University of Southern California, Department of Biological Sciences
Sarah Pierce
Senior Research Associate, King lab, University of Washington
Yan Qi
Assistant Professor of Biology, Amherst College
Brenda Reinhart
Research Associate, Roland Martin lab, University of Zurich
Christian Riedel
Assistant Professor, European Research Institute Biology of Aging, Groningen Netherlands
Holli Rowedder
Biology Department, Boston College
Sascha Russel
Teaching fellow, Harvard University
Ilya Ruvinsky
Assistant Professor, Dept. Ecology and Evolution, University of Chicago
Buck Samuel
Baylor College of Medicine, Department of Molecular Virology and Microbiology
Andrew Samuelson
Assistant Professor, University of Rochester
Gisela Sandoval
Child Psychiatry, Stanford University
Zhen Shi
Postdoctoral fellow, Maria Barna lab, Stanford
David Shore
Clark and Elbing, patent law
Frank Slack
Professor, Biology Dept., Yale University
Ann Sluder
Director of Biochemistry, Scynexis, Research Triangle Park
Alex Soukas
Assistant Professor, Harvard Medical School/MGH
Ji Ying Sze
Associate Professor of Pharmacology, Albert Einstein School of Medicine
Yuval Tabach
Assistant Professor at Hebrew University Hadassah Hospital (Israel)
Heidi Tissenbaum
Associate Professor, University of Massachusetts at Worcester
Duo Wang
Strategic Decisions Group, Boulder, CO
Meng Wang
Assistant Professor, Baylor University School of Medicine
Matthew Webber
Bruce Wightman
Professor of Biology, Muhlenberg College
Catherine Wolkow
Senior Scientist, Worm Atlas, New York
Xiaoyun Wu
Research Scientist, Broad Institute
Chi Zhang
Research scientist, Warp Drive Pharmaceuticals, Cambridge, MA
Xinrui Zhang
Case Western University, Cleveland, OH

Research

Gary Ruvkun is a professor of genetics at Harvard Medical School. Dr. Ruvkun is a graduate of UC Berkeley (AB, Biophysics,1973) and Harvard (PhD Biophysics, 1982).

Dr. Ruvkun’s honors and awards include the Rosenstiel Award from Brandeis University (with Victor Ambros, Andy Fire, and Craig Mello), the Warren Triennial Prize from Massachusetts General Hospital (with Victor Ambros), the Benjamin Franklin Medal from the Franklin Institute (with Victor Ambros and David Baulcombe), the Gairdner International Award from the Gairdner Foundation of Canada (with Victor Ambros), the Albert Lasker Award for Basic Medical Research (with Victor Ambros and David Baulcombe), the Louisa Horwitz Prize from Columbia University (with Victor Ambros), the Shaul and Meira Massry Prize (with Victor Ambros), the Dan David Prize for Aging research (with Cynthia Kenyon), the Ipsen Foundation Longevity Prize, the Wolf Prize in Medicine (with Victor Ambros and Nahum Sonenberg), the Gruber Genetics Prize (with Victor Ambros), the Breakthrough Prize in Life Sciences (with Victor Ambros), the National Academy of Sciences, the Institute of Medicine, and the American Academy of Arts and Sciences.

Curriculum Vitae

Developing a DNA detector for life detection on Mars

Searching for life beyond Earth is a major element of NASA’s missions and activities, many of which have focused on Mars as a world where life could have once existed and may still survive. Life on Mars, if it exists, may share a common ancestry with life on Earth derived from meteoritic transfer of microbes between the planets. We are building an instrument to test this hypothesis in-situ on Mars by isolating, detecting, and sequencing nucleic acids (RNA or DNA), the building blocks of all known life. We aim to develop a fully automated compact, portable version of this instrument, the Search for Extra-Terrestrial Genomes (SETG). It is also possible that RNA or DNA-based life may have arisen independently beyond Earth, an idea supported by the potential universality of biochemistry and the identification of amino acids, metabolic precursors, and nucleobases within meteorites, and ribose precursors in interstellar space. By isolating, detecting, and sequencing nucleic acids, SETG can analyze the genome or gene expression of any DNA- or RNA-based organism on Earth, Mars, or beyond.

The potential for common ancestry of life on Earth and Mars

An intense period of impact events called the Late Heavy Bombardment (LHB) occurred around 4.1-3.8 Gya, likely as a result of the inward migration of the giant planets. These impacts generated significant meteoritic exchange between Earth and Mars25, around 100x higher flux from Mars to Earth than vice-versa7. In the late 1990s, a series of theoretical studies demonstrated that Martian meteorites were transferred to the Earth at shortened time scales and with higher fluxes than previously believed, delivering around one billion tons of meteoric debris, representing 7.5% of all Martian meteorites. Within this collection, numerous meteorites would have been delivered on time scales of decades to thousands of years. Several dozen SNC meteorites of Martian origin have been discovered here on Earth, and magnetic and thermochronological analyses indicate that 20% of Martian meteorites have only experienced mild heating (<100ºC, below sterilization temperatures) during ejection and impact. Recent studies confirm the ability of bacterial spores to survive hypervelocity impacts. Once life had evolved on one of the planets, the rate of material transfer makes it plausible that the adjacent planet could “catch” life rather than independently evolving it.

The search for RNA or DNA beyond Mars

Recent discoveries of nucleic acids or their precursors within meteorites and in interstellar space22 could steer the development of life towards these biomolecules. Thus, it makes sense to search for RNA or DNA-based life within potential habitable zones even outside the context of meteoritic exchange, such as the probable liquid water oceans beneath Europa and Enceladus and possibly Titan. Given the possibility of shared ancestry between life on Earth and Mars, if it exists, and the potential for RNA or DNA-based life elsewhere, searching for life as we do know it is a critical part of any comprehensive life detection approach. Strategies such as detection of organic molecules (amino acids, individual nucleobases), molecular chirality, putative metabolic activity, specific protein modules, while valuable, either lack specificity or sensitivity. In contrast, SETG is extremely sensitive (down to single molecule) but does not sacrifice specificity: there are no known natural abiological routes to RNA or DNA sequences of nontrivial length.

Sequence data has redefined what we know about the nature and extent of life39. In addition to revealing that all known life (with the possible exception of viruses) is descended from a common ancestor, such sequences have revealed entirely new high-level taxa. All known (non-viral) life forms share about 500 “universal genes” including the ribosomal RNAs, regions of which have changed very little over the past 3-4 billion years45. For example, within the ~1500 nucleotides of the 16S rRNA gene (18S in eukaryotes), there are multiple 15-20 nucleotide segments that are nearly identical in all known organisms46 because these regions are involved in regulating the genetic code47, the degenerate mapping of nucleobase triplets to amino acids. The ribosomal sequences are the gold standard for identifying and classifying diverse microbes. The centrality of these RNAs led to the idea that an RNA world may have preceded the DNA world, which provides an incentive for SETG to target both RNA and DNA.

SETG can target a specific gene or gene region between two known primer sequences using polymerase chain reaction or target all DNA or RNA (RNA is first reverse transcribed to DNA). Sequencing any DNA molecule is important for targeting putative nucleic acid-based Martian life, where the extent of ancestry is unknown. Sequence data can then be used to place an organism on the tree of life, identify its closest known relatives, or distinguish between closely related species, through an analysis of similarity to other sequences. For example, the Green Genes database contains >700,000 near full-length 16S ribosomal sequences.

Library Generation for Sequencing

Aside from single-molecule sequencing approaches, which are impractical for our application due to their complexity, sequencing requires generating thousands to millions of identical copies of the same DNA molecule. This requires having known sequences at the ends of the molecule that can be targeted, which is entirely straightforward when targeting a particular gene with primers. When targeting any DNA molecule, one must fragment the DNA to a desired size, add known ends, amplify to get a clonal product, and sequence.

Nucleic Acid Amplification and Detection

We originally focused on targeting the most highly conserved regions within genes shared by all known life: We analyzed whole genomes, and identified regions in the ribosomal 16S and 23S genes as the most conserved, followed by transfer RNAs14. Notably, these regions are all part of the RNA system that regulates the genetic code47. Our prototype amplification/detection module allowed us to amplify up to 8 samples on a microfluidic chip, with each sample amplified in 384 wells each of 1 nanoliter in volume. Average power during thermal cycling was ~25W. This design would facilitate selection of a particular clonal product to use for sequencing. We also developed short primers to precisely target highly conserved gene regions. In-silico and in-vitro assessment of these primers suggests they can amplify the vast majority of known organisms and can detect more biodiversity than longer primers55. However, no primer set was found to be truly universal, and the short primers suffer from poor PCR efficiency. Thus, the capability to amplify any RNA or DNA molecule is critical in order to detect potentially divergent Mars organisms that have been isolated from Earth organisms for >3.5 billion years. Fortunately, sequencing of RNA in microbes reveals that >90% of microbial RNA is ribosomal; thus we can directly target these highly conserved genes by sequencing RNA, and use these sequences to evaluate potential ancestral relationships.

Massively parallel sequencing

Until recently, sequencing instruments have been large, heavy, complex, and required specialized reagents and sensitive optics. For this reason, we had originally proposed to develop a single channel sequencer, which would have allowed us to keep the size small and use non-imaging optics to do pyrosequencing, at the expense of a very limited sequencing capability. However, massively parallel sequencing is now feasible for SETG, based on the technology commercialized by Ion Torrent (Fig.): a small standard semiconductor chip that enables concurrent sequencing in millions of wells, requires no imaging or optics, and is extremely small, fast, and robust. In addition, massively parallel sequencing enables us to dramatically simplify our earlier design, eliminating imaging optics and reducing fluidic complexity. Consider one well on the sequencing chip, occupied by a bead covered in a single clonal DNA molecule. When a matching nucleotide flows by, a polymerase enzyme sitting on the DNA will incorporates the nucleotide into the 3’ end of a growing double-stranded DNA molecule, releasing a hydrogen ion. When this happens concurrently on ~106 identical molecules, the resulting transient change in pH is detected as a change in the source voltage V. By flowing different nucleotides one by one, and looking for transients, the target sequence can be determined in each of the occupied wells. By fitting these transients to a model of nucleotide incorporation, each base can be called and scored for quality. We are collaborating with Ion Torrent founder Jonathan Rothberg , who has interest in space applications of sequencing. Rothberg is well known for founding 454, where he pioneered massively parallel sequencing. Specific contributions by Ion Torrent will include providing custom sequencing chips, and miniaturization of the supporting electronics. Ion Torrent technology is by far the most practical technology for space applications.

Our concept for SETG is an instrument 3 kg in mass with a volume a bit more than a typical laptop, peak power < 30W, and average power of ~10W during a run. We envision SETG and other life detection instruments utilizing a common sample collection system such as the one on the Mars Science Lab. For our integrated instrument concept (Fig. 6), a person would load a sample into a single-use cartridge, which would include all fluidics, freeze dried reagents, buffer, other small non-reusable components, and the sequencing chip. This cartridge would have electrical and pressure interfaces to a hardware module, which would be capable of operating autonomously, but could be controlled or monitored via a computer or smart phone. The hardware module itself will have little or no user interface other than power, data, and controls or interlocks for safety or convenience. The process will be completely automated once a user loads a sample, seals the sample inlet, and initiates a run.

Fig. Integrated instrument concept. All fluidics are contained within a single cartridge to prevent cross contamination between runs. To enable processing of multiple samples on Mars, this cartridge could be replicated or split to enable common use of some components: for example, multiple cartridges making library-ready DNA could be sequenced in a common sequencing module.

Lifespan and Metabolism

During the last 15 years, the insulin/IGF-1/daf-2 signaling pathway has been shown to be the most potent regulator of lifespan in C. elegans. Signaling from DAF-2 is mediated through the AGE-1 phosphatidylinositol 3-kinase (PI3K), PDK-1, and AKT-1/2 kinases, to antagonize DAF-16, orthologous to human FoxO, a forkhead transcription factor. The function of this pathway in mediating metabolism and aging is conserved in C. elegans, Drosophila, and mammals. Because so much of the insulin signaling pathway is conserved, the new components we discover in C. elegans will have broad relevance to mammalian insulin signaling and longevity control.

Genetic analysis in C. elegans continues to identify components of the pathway that are likely to reveal human variation in insulin-like signaling, with medical significance for diabetes and the understanding of how insulin signaling and analogous hormonal pathways couple chronological age to many late onset diseases. Using RNAi to screen for defects in daf-2 pathway mediated longevity regulation, we identified a comprehensive genetic network necessary for the longevity response to low daf-2 insulin/IGF1 signaling (9). Similarly, our proteomic analysis of insulin signaling components has identified other new and unstudied candidate genes to act in insulin signaling. The use of RNAi screens and proteomics in C. elegans is opportune for two reasons: first, the tissues where insulin signaling is key for metabolic control has changed dramatically over the past decade. No longer is an exploration of insulin signaling only in the liver or muscle or even pancreas definitive. Neural and adipose centers of insulin signaling have emerged. We have identified new protein components of insulin signaling from whole animal extracts, and our RNAi screens are done in the whole animal, so insulin signaling across tissues is surveyed. This is unlike mammalian insulin signaling functional genomics which may assay for insulin responses in tissue culture, but not in the physiological context of a whole organism. Aging and diabetes may be more physiological and endocrine, not easily modeled in cell culture. In this way, the C. elegans insulin signaling genetic system is better model system for human aging and human diabetes than human cell culture.

Gene activities that mediate the longevity regulation by C. elegans insulin-like signaling. Samuelson, A. V. , Carr, C. E., and G. Ruvkun. 2007. Gene activities that mediate increased lifespan of C. elegans insulin-like signaling mutants. Genes and Development 21:2976-94.

Genetic and RNA interference screens for lifespan regulatory genes have revealed that the daf-2 insulin-like signaling pathway plays a major role in C. elegans metablism and longevity. This pathway converges on the DAF-16 transcription factor to regulate aging by controlling the expression of a large number of genes. We conducted a genome-wide RNA interference screen to identify genes necessary for daf-2 mutants to survive nearly twice as long as wild type, and identified approximately 200 gene inactivations that shorten daf-2 lifespan to near that of wild type but have much more minor impact when inactivated in wild type. Of the gene inactivations identified in our screen, most enriched are genes annotated to mediate vesicle sorting. For example, compared to loss of daf-16, inactivation of Y65B4A.3 caused the greatest suppression of daf-2 phenotypes. Y65B4A.3 is homologous to human charged multivesicular body protein 6 and the myristolyated subunit of yeast ESCRT-III, the endosomal sorting complex required for transport of transmembrane proteins into the multivesicular body pathway to the lysosomal/vacuolar lumen. Sixteen other endocytosis/vesicular trafficking related genes also suppressed daf-2. One of the responses to a decline in daf-2 is dramatic upregulation of the DAF-16 target gene sod-3, a manganese superoxide dismutase. Thirty-four gene inactivations suppressed the daf-2-dependent induction of sod-3 expression, including smk-1/ protein phosphatase regulatory subunit, mag-1/exon junction complex component, F28D1.9/fatty acid transporter, and cua-1/cation transporter.

Proteomic exploration of the insulin signaling pathway (not yet published)

In the absence of insulin-like signaling, DAF-16/FoxO binds to the promoters of numerous metabolic and stress response genes and can either activate or repress their transcription by RNA Pol II. Such transcriptional outputs cause enhanced stress resistance and a change to fat storage metabolism, and enhanced longevity. Using a proteomic approach, we have identified dozens of candidate protein interactors with insulin-signaling components. We constructed functional translational fusions to epitope tags for the transcription factor FOXO/DAF-16 and one of its activators, SMK-1. FOXO/DAF-16 mediates the transcriptional output of insulin/IGF signaling. SMK-1 is predicted to be a regulatory subunit of protein phosphatase 4 and has genetically been shown to promote DAF-16 activity. We integrated the fusion protein constructs into the C. elegans genome and to study DAF-16/FOXO in variable states of activation, we used three different strain backgrounds: wild type, daf-2(e1370) a strong kinase domain mutant with low insulin like signaling, and daf-18(mg198)/PTEN phosphatase null allele with constitutively high insulin like signaling. Epitope-tagged DAF-16/FOXO from each strain grown in large quantity (tens of liters of growth medium and tens of ml of packed worms) was purified by immunoprecipitation from each of these strains. Co-purifying potential physical interactors of the fusion proteins were identified by mass spectrometry (LC-MS/MS).

In order to determine the background of these purifications, parallel control purifications from worms lacking the fusion proteins were performed. Many of the FOXO/DAF-16 binding partners have chromatin or stress response related annotations. To test their function in daf-2 regulation of lifespan, we inactivated each corresponding gene by RNAi and surveyed phenotypes expected for either enhancement or loss of DAF-16/FOXO activity. We tested each gene inactivation in wild type animals for an extension of lifespan, enhanced resistance to heat or oxidative stress or inappropriate expression of the DAF-16/FOXO-activated gene sod-3. Conversely, we tested in daf-2/insulin receptor mutant animals for suppression of extended lifespan, suppression of resistance to heat or oxidative stress or an inability to overexpress sod-3. Of 89 binding partners tested, 65% showed significant lifespan or stress resistance phenotypes in at least one and 32% in multiple assays. The genes identified by this screen are highly enriched for acting in the longevity pathway: 18 % of the DAF-16/FoxO binding partners are required for lifespan extension in daf-2(e1370), while a genome-wide screen for the same phenotype had a discovery rate near 1%.

Lifespan regulation by evolutionarily conserved genes essential for viability Curran, S. P. and G. Ruvkun. 2007. PloS Genetics 3(4):e56

We have discovered that other arrest points induced by gene inactivations of core cellular components such as the ribosome or mitochondrion or cytoskeleton may be as highly regulated and as key to lifespan regulation as the insulin regulated dauer arrest point. In common with the dauer arrest point, these other arrest points are induced by environmental inputs, for example starvation or natural products that target conserved cellular components such as the mitochondrion or the ribosome. Our analysis suggests that the integrity of these core cellular components are assessed, either in cells that tend to be the most exposed to the environment, or in all cells, and that a signaling pathway to endocrine control of development and reproduction may operate.

To reveal this essential pathway signaling system, we screened the 2700 gene inactivations that cause larval arrest or reproductive arrest in C. elegans for increased adult lifespan by initiating the gene knockdown once the animal had reached adulthood, thus bypassing any developmental abnormalities. We identified 64 genes that can extend lifespan when inactivated post-developmentally. More than 90% of the genes we identified were conserved from yeast to humans. Our yield of 64 gene inactivations out of 2700 tested (~2.4%) is a four-fold increased yield than the previous 89 gene inactivations out of 16,000 screened (~0.6%), and a higher proportion of the gene inactivations cause large increases in longevity.

To classify the pathways represented by these new genes, we performed secondary assays: DAF-16 localization, sod-3 expression, arrested larval survival, suppression of polyglutamine aggregation, and aberrant fat metabolism and clustered the genes by the phenotypes observed. Our analysis placed some of these longevity genes within the insulin-signaling pathway, while others were independent of this pathway.

Stress resistance may contribute directly to extended longevity, and is co-regulated with longevity. We have observed a panel of a dozen stress-responsive GFP fusions in strains experiencing gene inactivations corresponding to a library of ~200 lifespan-extending RNAi clones. We tested a heat induced GFP fusion, hsp-16::GFP, an ER stress induced gene, hsp-4::GFP, and two mitochondrial stress induced genes, hsp-6::GFP and gst-4::GFP. Most of the longevity inducing gene inactivations activated one or more stress reporter genes and the pattern of stress reporter activation was characteristic for ribomsomal inactivation for example compared to mitochondrial inactivation.

The integrity of these core cellular components may be assessed in cells that tend to be the most exposed to the environment, and a signaling pathway to endocrine control of development and reproduction may operate. For example, we have found that there are sentinel signaling cells: the intestine where microbes are first encountered in an intimate way and sensory neurons that might “probe” the antibiotic environment before the rest of the cells of the animal are inhibited. We used a collection of strains in which gene knockdowns can be accomplished within a single, restricted tissue using the same technique of feeding animals bacteria expressing dsRNA against target genes. The strain backgrounds used for these experiments are fully defective for RNAi because they lack the necessary gene (either rde-1 or sid-1). Only the tissue(s) in which gene function is inactivated by RNAi of essential genes are those in which the tissue-specific promoters rescue rde-1 or sid-1 expression is rescued, in neurons or intestine or muscle. In addition, we used a mutant strain proficient for RNAi in the germline but not in somatic tissue (rrf-1) due to differing mechanisms of RNAi in these tissue types. Inactivation of core cell components in each tissue tested – the hypodermis, the intestine, the gonad and body wall muscle – is competent to trigger similar responses to essential gene inactivation as inactivation of these genes in all tissues. This suggests the existence of an endocrine system for response to essential gene inactivation. Similar endocrine outputs from mitochondrial gene inactivation have been found by the Dillin lab (12).

The developmental arrests induced by these gene inactivations and the longevity induction may be a “programmed” response to a deficiency in a key function, and active signaling programs may mediate the arrest point, as a sort of “developmental checkpoint”. Such an arrest program interpretation depends on two key attributes: 1. The arrest should be reversible. We have observed that when drug is removed in many cases, the arrest is reversible. 2. There should be mutations that disable the arrest program. This is also established below.

Stress decoupled mutants shorten the lifespan of insulin signaling and other long lived mutants (not published)

A common theme to many of the cellular components that induce increased longevity when inactivated is that many are targets of antibiotics produced by a range of fungi and microbes that nematodes encounter in the environment. We hypothesize that as a larvae or adult enters an environment with an antibiotic, there may be signaling pathways that detect, for example, antibiotic-induced ribosomal deficiency to trigger cessation of reproductive developmental trajectory, arrest at a particular developmental point, as well as longevity enhancing pathways and behavioral aversion programs to allow the arrested animal to escape and survive long enough to reanimate reproduction and be alive for that joyous experience. The induced stress adaptation and survival pathways would ensure that the animal could escape the antibiotic and resume reproductive development in a less toxic environment or feeding on less toxic microbes. Inhibition of translation by RNAi of translation factors may mimic the ribosomal function deficiency induced by antibiotics in the normal C. elegans ecosystem, and trigger the physiological response of developmental arrest and cessation of reproduction and initiation of longevity programs in the arrested larvae or adults. Exposure of C. elegans to many of these drugs cause similar developmental arrest and a modest increase in lifespan, but the drugs are not as potent as the gene inactivation by RNAi. This may be because drug detoxification is highly evolved —-they induce a variety of detoxification pathways that may be more effective on the small molecule drugs than on gene inactivation by RNAi.

The same stress reporter genes activated by the longevity inducing gene inactivations are also induced by the drugs. Tunicamycin is a natural product the bacterium Streptomyces Iysosuperficus; it inhibits ER N-linked glycosylation and strongly induces HSP-4, a component of the ER unfolded protein response. Antimycin is a natural product Streptomyces and inhibits the mitochondrial electon transport complex 3 by binding the cytochrome b subunit and induces hsp-6, a component of the mitochondrial unfold protein response.

We screened for new gene inactivations that cause a failure to induce the ER or mitochondrial or other stress GFP fusion genes under drug or stressed conditions—-these gene inactivations may cause animals to be “blind” to mitochondrial or ribosomal dysfunction, and therefore not induce these GFP fusion genes. We then asked if a failure to induce these genes reflects a general decoupling of the surveillance of the ribosome for example to the induction of longevity normally induced by essential gene inactivations. In this way, we will discern the signaling pathway, both within cells and between cells for decrements in core cellular functions.

So far from these pilot screens, we have identified 30 gene inactivations that fail to activate the drug induced reporter genes. If the cytoprotective pathways used as stress reporters in the studies above are normally induced by mutants or gene inactivations that confer increased longevity and are part of the program for increased longevity, a decoupling of their induction might shorten the lifespan of long lived mutants more than wild type. We tested whether the 30 gene inactivations with defects in cytoprotective gene induction also abrogated the increase in lifespan induced by mitochondrial dysfunction, reduced feeding, or disruption of insulin signaling. In these experiments, 12 of 30 gene inactivations tested abrogate 2/3 or more of the lifespan extension observed in eat-2, isp-1 and/or daf-2 mutants. While dcp-66, pas-3 and arf-3 exert their largest suppression of lifespan in isp-1, inactivation of cpf-2, wnk-1 and nekl-2 are most potent in the eat-2 mutant. phi-50, ima-3, gob-1, ufd-1, let-70, and elt-2 are critical to lifespan extension in both the isp-1 and eat-2 mutants. Two of these, phi-50 and ima-3, also reduce the lifespan of daf-2 mutants by more than 2/3. These gene inactivations are analogous to the ceh-23 suppression of mitochondrial mutant lifespan increase (16).

A soma-to-germline transformation phenotype in long-lived C. elegans mutants (Curran et al, Nature 459: 1079-84)

Genomic instability of somatic cells is a hallmark of increased age among most organisms. Protection of the germline on the other hand is an evolutionarily conserved trait. We uncovered a soma-to-germline transfomation phenotype among Caenorhabditis elegans longevity mutants. Mutations that inhibit insulin-like signaling cause misexpression of a germline restricted gene, pgl-1::gfp in the intestine and hypodermis. Mutants in the synMuvB class of genes are Eri and display somatic misexpression of the normally germline-limited P-granule component PGL-1. We monitored a PGL-1::GFP protein fusion in daf-2 or age-1 mutant strains. pie-1 and pgl-1 are exclusively expressed in the germline of wild type animals. Decreased insulin-signaling caused strong misexpression of PGL-1:GFP in hypodermal and intestinal somatic tissues of dauers and late larval stage animals. By qPCR, we could verify strong up-regulation of the P granule components pgl-1, pgl-2, and pgl-3 in the soma of daf-2 and age-1 mutants. This misexpression of germline components was strongly suppressed by mutations in the FoxO transcription factor DAF-16, the transcriptional output that is repressed by the insulin-like signaling pathway.

The misexpression of germline markers suggests that the somatic cells of an insulin-signaling mutant are more germline-like. Germline-transformed somatic cells, like germ cells, may engage additional protective pathways that prevent or slow genomic destabilization; the effect of which could cause an increased ability to respond to stress and extend lifespan. We tested the ability of daf-2 mutants to protect somatic tissues from genomic instability by feeding a sublibrary of RNAi clones previously shown to induced somatic nuclear DNA mutation. RNAi targeting rpa-2, srxa-6/dnJ-25, and F49E12.6 causes an increased rate of somatic mutation. These gene inactivations cause nuclear DNA damage as measured by the somatic expression of a normally out-of-frame lacZ transgene. Expression is induced following a nucleotide insertion/deletion event that places the lacZ gene in-frame. After feeding these RNAi clones, X-gal staining of animals carrying this transgene in the daf-2(e1368) background displayed reduced lacZ expression in somatic cells compared to animals harboring the transgene alone, suggesting a reduction in genomic instability in the somatic cells of the insulin-like signaling mutants.

If the misexpression of germline genes in somatic tissues contributes to the longevity phenotype of insulin signaling mutants then removal of the genes by RNAi should suppress the longevity phenotype. To test this we used RNAi to ablate the misexpressed germline transcripts in daf-2 mutant animals. Depletion of these misexpressed germline genes in the daf-2 mutant reduced the lifespan of these normally long-lived animals but not wild type animals. The enhanced RNAi phenotype of synMuvB mutations is suppressed by loss of function mutations in chromatin remodeling ATPase, isw-1 and the SET domain containing protein, mes-4. Similarly, RNAi towards isw-1 and mes-4 could partially suppress the enhanced longevity of daf-2(e1368).

We tested whether any of the other genes identified in our post-developmental RNAi screen to extend lifespan also displayed somatic cell to germline cell fate transformations by tracking the expression of endogenous PGL-1. PGL-1 is normally restricted to the germline where it forms perinuclear punctate structures around mitotic and meiotic germcells. RNAi clones targeting two components of the cytosolic chaperonin complex cct-4 and cct-6 also caused somatic expression of PGL-1. The somatic PGL-1 protein localized to punctate perinuclear rings in hypodermal cells and also produced intestinal cytoplasmic granules.

Germ cells employ protective mechanisms to ensure genetic integrity. The soma-to-germline transformation provides a potential mechanism for the observation that long-lived mutants exhibit resistance to genotoxic stress. We hypothesize that increased genomic stability in somatic cells may be an important pathway facilitating lifespan extension. It will be interesting to investigate if this is a broadly conserved mechanism given that protection of the germline is a shared trait across species.

We have discovered that the daf-2 insulin/IGF1 pathway also regulates the intensity of RNAi in C. elegans. Long-lived mutants in daf-2 and age-1 are enhanced for RNAi and misexpress the normally germline restricted gene P granule RNAi components in the intestine and hypodermis. Mutants in the synMuvB class of genes are also enhanced for RNAi (Eri) and display somatic misexpression of the normally germline-limited P-granule components. The misexpression of germline markers suggests that the somatic cells of an insulin-signaling mutant are more germline-like. Germline-transformed somatic cells, like germ cells, engage additional protective pathways that prevent or slow genomic destabilization to cause an increased ability to respond to stress and extend lifespan. The germline RNAi components that we study are universal to animals. The genes we will discover in this proposal that mediate the regulation of the P granule components are likely to be universal to all animals and to act in aging pathways of humans as well.

Our genetic analysis of aging in the reproductive system of C. elegans is thematically related to the regulation of germ cell pathways in somatic cells. We have found many gene inactivations affect reproductive senescence, some of which also affect organismal aging, some of which do not. Because the reproductive system in mammals is tightly coupled to an endocrine system that assesses nutritional and stress status, our genetic analysis of this system in C. elegans is likely to reveal previously unknown endocrine axes and signals.

Regulation of RNAi by the daf-2 insulin-like signaling pathway (published as Wang, D. and G. Ruvkun. 2004. The insulin pathway regulates RNAi in C. elegans. Cold Spring Harbor Symposium on Quantitative Biology, 69th edition, 69: 429-433.).

C.elegans strains with decreased insulin-like signaling have a more intense RNAi response than wild type. Such regulation of RNAi by this stress and longevity signaling pathway suggests a role in response to pathogens such as viruses. For example, mutants lacking age-1 show enhanced response to RNAi. age-1(mg305) responds to RNAi of lin-1 with a multiple vulva phenotype in 96% of the animals vs 0% on wild-type animals. Similarly, other RNAi of a range of histone genes is enhanced in age-1 strains. Northern analysis shows that after feeding his-44 dsRNA, his-44 mRNA level is significantly decreased in age-1(mg305), whereas no change is observed in wild type. A daf-2; daf-16 double mutant or an age-1; daf-16 double mutant do not show enhanced RNAi. These data suggest that insulin-like signaling normally inhibits RNAi via the DAF-16 transcriptional cascade. Dauer-constitutive mutants in other two pathways, daf-7/ TGF-b and daf-11/ guanylate cyclase are either unaffected or very weakly enhanced, in contrast to the daf-2 pathway mutations. Our model for this enhanced RNAi is that is the misexpression of germline specific RNAi factors that confers the enhanced somatic RNAi in insulin signaling mutants.

Lin-35/retinoblastoma regulation of somatic/germ line specification (Wang et al, Nature 436: 593-597 and unpublished date since that paper being written up now)

The Retinoblastoma (Rb) protein functions as a crucial transcriptional repressor in tumor suppression. In C. elegans, genes encoding Rb (lin-35), components of the core complex and the recruited chromatin factors belong to the synthetic multivulva B (synMuv B) gene class, which repress transcription during vulval development. A whole genome RNAi screen for synMuv suppressors identified mostly chromatin factors7, suggesting that, as in other organisms, the biological roles of lin-35/Rb in C. elegans are primarily chromatin regulation.

Mutations in lin-35/Rb lead to soma-to-germline transformation, similar to the daf-2 insulin signaling mutants. As in the case of the daf-2 mutant, lin-35 mutant worms misexpress the germline specific protein PGL-1 in somatic tissues8. Unlike daf-2 mutant animals, these ectopically expressed PGL-1 proteins form perinuclear granules, reminiscent of its natural P-granule localization in the germline9, indicating that additional germline specific genes may also be misexpressed. lin-35 mutant worms also misexpress germline-enriched genes in somatic cells. Therefore, loss of lin-35/Rb induces a germline state in the soma. PGL-1 misexpression in lin-35 and other synMuvB mutants can be suppressed by inactivating several Muv suppressing chromatin factors7, 13, 14 (mes-4, isw-1 and mrg-1). However, not all Muv suppressing-chromatin factors are PGL-1 suppressing7, suggesting that preventing soma-to-germline transformation is different from preventing Muv formation.

The added germline expression in the soma of lin-35 mutants likely contributes to some of their organismal phenotypes. This is particularly likely for the enhanced RNAi phenotype observed in lin-35 mutants, since many genes functioning in RNAi or related processes are predominantly expressed in the germline15. Indeed, several germline-enriched Argonaute genes are significantly misexpressed in the soma of these worms.

To examine the extent of germline transformation, we compared two microarray analyses performed on lin-35 mutant worms at the first larval stage (L1)10, 11. L1 worms contain a very minimum germline and thus mostly represent expression profiles in the somatic tissues. This analysis identified 295 genes that are commonly upregulated in both arrays. Among them, 141 genes (~50%) were normally germline-enriched as they were depleted in mutant worms lacking a germline15. Thus, germline genes are preferentially upregulated in lin-35 mutant worms, suggesting an extensive germline transformation in the soma.

To verify such a germline transformation in the soma, we measured germline-specific transcripts in the soma of lin-35 mutants. This was achieved by performing the experiments in a glp-4 mutant, which are defective in germline proliferation so only expression in somatic cells is monitored in whole animal RNA preps. We focused on 9 genes that are involved in small RNA function, including genes that encode germline-enriched RNAi factors and components of the germline-restricted P-granules. Real-time RT-PCR on isolated total RNA confirmed the upregulation for all 9 genes. This is shown Table 1.

We further characterized the function of the heterochromatin factors via a biochemical approach. We created transgenic lines that express GFP-tagged LIN-61/L3MBT and immunopurified LIN-61::GFP containing protein complex using anti-GFP antibody. Copurifying proteins were identified by mass spectrometry. The only synMuv B proteins that copurify were LIN-13 and HPL-2/HP1, known synMuvB genes, suggesting the existence of a separate complex formed by synMuv B heterochromatin proteins.

The extensive misexpression of germline genes suggests that germline transformation may involve the activation of one or more master regulators that trigger subsequent cascades of misexpression. Inactivation of mes-4, encoding a histone H3K36 dimethyltransferase21, suppress multiple phenotypes of lin-35 mutants, including Muv and PGL-1 misexpression. We found that the somatic misexpression of 4 of the 9 genes in the pilot set depends on mes-4 (Table 1, last 4 columns), showing that it is a master regulator.

Genome-wide RNAi screening for late reproductivity genes (not yet published)

There is a correlation between late fertility and longevity in many species. Both daf-2 mutants with decreased insulin like signaling and tph-1 mutants deficient in serotonin signaling that act upstream of daf-2 cause late fertility. We developed a simple genetic selection for late reproduction mutants. We use the sqt-3 (e2117) temperature sensitive collagen mutant to kill off the early clutch of embryos laid at the non permissive temperature of 25 degrees. sqt-3 encodes a cuticle collagen, the temperature sensitive allele (e2117) of which is embryonic lethal at 26℃. sqt-3 (e2117) mutants cease progeny production completely and undergo reproductive senescence after day 4 of adulthood at 26℃. To screen for mutants with delayed reproductive senescence, sqt-3 (e2117) mutant animals were fed a library of RNAi clones to test each of 20,000 gene inactivations, were grown at 26℃ for 5 days, during which all F3 progeny are lethal due to the collagen mutation. This lethality is not reversible by lowering the temperature. After the mostly post reproductive 5 day old adults are shifted to 15 degrees, wild-type reproductively senescenced worms will not produce any progeny, while the mutants or gene inactivations with late reproductivity still generate progeny. The background that we must distinguish are gene inactivations that induce greater production of sperm, which naturally limit brood size, and gene inactivations that generally slow down metabolism and development, essentially making the rate of living during the 5 day senescence period a shorter period in biological time. Our intitial controls for this are to measure brood size relative to control; those strains with a larger than normal brood size are suspected to have increased sperm production. This could also be due to increased lifespan of sperm, not an uninteresting topic. Our control for slower metabolism is to record the developmental time from L1 to adult; those gene inactivations with normal developmental time, and normal brood size are our top candidates. We have screened the entire genome based on this experimental design. Totally, 32 genes have been identified from these primary screens. Most but not all of these hits do not affect growth rate or brood size. The hit rate is around 1%. One recent paper from the Murphy lab found that the daf-4 TGF beta pathway receptor affects C. elegans reproductive lifespan 155. We did retrieve daf-3 in our screen, endorsing this finding. But many of our hits are much better than daf-3, so we believe that our analysis will be more thorough.

Our Science

Small RNA pathways regulate gene expression, chromatin structure and defense against invading elements such as transposons and viruses. Small RNA deep sequencing experiments have shown that about half of all genes produce endogenous siRNAs suggesting that this regulatory axis controls a wide range of gene activities. siRNAs are incorporated into effector complexes, comprised of an Argonaute protein and accessory factors, which direct silencing of complementary RNAs and in certain species, such as C. elegans, recruit RNA-dependent RNA polymerases (RdRPs) to the target, resulting in siRNA amplification. All small RNAs act sequence-specifically through base pairing with their target mRNA, but the outcome of the small RNA:target interaction can vary from suppression of transcription to mRNA degradation or translational repression and this is likely governed in part by the specific Argonaute cofactor and in part by other coupled proteins that mediate the sorting of these small RNAs within the cell and between cells. Much remains to be learned about miRNAs and siRNAs and how they silence gene expression. There are hints that they sequester target mRNAs in P bodies and other subcellular structures, and constitute elements in an RNA trafficking system. There are hints of extracellular movement of small RNAs, perhaps constituting a shadow endocrine system. The genes identified are likely to be key factors in the function of small RNAs and their identification may enable more potent RNAi based drug development.

Our work follows up on our past full genome screens for candidate cofactor genes and our development of molecular signatures for small RNA defects using deep sequencing. The gene lists identify the key elements in the choreography of small RNAs in C. elegans. Because the genes are nearly all conserved across phylogeny, it is likely that the rules we work out in C. elegans will apply generally. Our goals are to use our deep sequencing analyses and new reporter genes for endogenous siRNAs and miRNAs to transform these lists of about 500 genes into coherent pathways of RNA processing, trafficking and presentation to the genome. We expect that the implications of the work will be to discover to new elements in how miRNAs and siRNAs are generated and engage cellular proteins to in turn engage their targets, as well as the discovery of other small RNA pathways from our analysis of the genes we have identified.

There have been 2 general areas of small RNA biology that we have developed: the mechanisms of exogenous and endogenous RNAi and the mechanism of miRNA regulation of target mRNAs.

The exonuclease ERI-1 plays a conserved dual role in RNA interference and ribosomal 5.8S RNA processing. Gabel, H. W. and G. Ruvkun. 2008. Nat Struct Mol Biol. 15: 531-3.

In C. elegans and fungi and plants, RNA dependent RNA polymerases amplify primary siRNAs to produce a more robust response. This two stage amplifier with feedforward amplification, like PCR, can amplify noise, so as in good electronic design, is likely to be attenuated. To identify negative regulators of C. elegans RNAi, we carried out a genetic screen for mutants that show an enhanced RNAi (Eri) phenotype. Mutants in eri-1 display an enhanced RNA interference (Eri) phenotype in which dsRNAs that are ineffective in silencing target mRNAs in wild-type trigger robust silencing in an eri-1 null mutant. eri-1 has a conserved endogenous function that was previously unsuspected: the 5.8s RNA inappropriately retains 1 to 4 nucleotides of its precursor in the eri-1 mutant compared to wild type. The homolog of ERI-1 in the fission yeast S. pombe also negatively regulates RNAi and we also observed that the 5.8S rRNA in the S. pombe eri1∆ mutant has 2 to 8 additional 3’ nucleotides. The Rao group at Harvard also discovered that mammalian ERI-1 targets the 5.8S RNA analogously (1). Thus ERI-1 plays a conserved dual role in the RNAi and rRNA processing pathways.

Trans-splicing in C. elegans generates the negative RNAi regulator ERI-6/7. Fischer, S.E.J., M. D. Butler, Q. Pan and G. Ruvkun 2008, Nature 455: 491-6.

Two other mutants from the enhanced RNAi genetic screen, eri-6 and eri-7 map to two adjacent ORFs present on opposite strands but failed to complement. The syntenic region in C. briggsae revealed that eri-6 and eri-7 constitute one gene on one strand in this species. The eri-6/7 protein is a member of the superfamily I DNA and RNA helicases. The divergently transcribed RNAs for eri-6 and eri-7 are assembled in C. elegans into one mRNA by trans-splicing. A 930 bp repeat 3’ to eri-6 and 5’ to eri-7 mediates this trans-splicing. We proved this by the detection adenosine to inosine editing of the proposed RNA duplex mediated by an adenosine deaminase (ADAR). A strain with mutations in both adenosine deaminase genes enhances transgene silencing, suggesting that editing of dsRNAs normally inhibits RNAi. One model for the increase in transgene silencing the absence of these ADARs is that the dsRNA element in eri-6/7 is a better target for the RNAi pathway when it is not edited, thus decreasing eri-6/7 gene activity.

The ERI-6/7 Helicase Acts at the First Stage of an siRNA Amplification Pathway that Targets Recent Gene Duplications (Fischer, SEJ., TA Montgomery, C Zhang, N Fahlgren, A Hwang, CM Sullivan, JC Carrington and G Ruvkun 2011, PloS Genetics 7(11): e1002369.

To characterize the role of eri-6/7 in endogenous siRNA pathways, we compared the small RNA profiles of eri-6/7 mutants as well as ergo-1 and eri-1 mutants to wild type. siRNAs corresponding to about 80 target genes were depleted in eri-6/7 mutants and the corresponding mRNA levels were up-regulated. The genes targeted by eri-6/7 show extensive nucleotide homology to each other but are not conserved between C. elegans and C. briggsae. These genes also have few introns, supporting the model that these genes have recently been acquired by C. elegans, perhaps from viruses. ERGO-1/ERI-6/7 pathway may constitute elements of such a viral surveillance pathway that extends beyond the initial infection.

An endogenous siRNA sensor reporter gene to easily assay the production or response to endogenous C. elegans siRNAs Montgomery TA, E. Rim, N Fahlgren, C Sullivan, JC Carrington, and G. Ruvkun.).

We developed a GFP based sensor for endogenous siRNA activity in C. elegans. 22G siR-1 is derived from a cluster of 22G siRNAs on the X chromosome that are dependent on embryonically expressed upstream ERGO-1 generated 26G siRNAs. The siRNA sensor, ubl-1::gfp-siR-1-sensor, contains a single engineered perfect duplex target site for an abundant endogenous siRNA, 22G siR-1, embedded in the 3’ UTR of ubiquitin-like1 (ubl-1) and expressed under the control of the ubl-1 promoter, which is active in all tissues throughout development (Figure 1). A control construct ubl-1::gfp, lacks the siRNA target site. Each construct was introduced into C. elegans using Mos1-mediated single copy insertion. GFP expression was ubiquitous in C. elegans containing the control, which lacks the 22G siR-1 target site, but was nearly absent in C. elegans containing the reporter with the 22G siR-1 sensor element in the 3’ UTR. The silencing of the siRNA sensor is suppressed by ergo-1 or eri-6/7 RNAi.

To determine if siRNAs trigger production of siRNAs in the genomic vicinity of the initial target site, we deep sequenced small RNAs from C. elegans containing either the ubl-1::gfp or ubl-1::gfp-siR-1-sensor transgene. 22G siRNAs were uniformly distributed across both transgenes and were derived exclusively from coding and vector sequence and not from the ubl-1 5’ and 3’ untranslated regions (Figure 1CD). A large peak was observed at the siRNA target site of the sensor, it is 22G siR-1 that may originate from the endogenous X-cluster siRNA locus, as the levels of 22G siR-1 were identical between control- and siRNA sensor-transgenic C. elegans. But this abundant siRNA does not attract more siRNAs to the region. These results suggest that, unlike primary exogenous siRNAs and endogenous 26G siRNAs, 22G siRNAs do not trigger siRNA amplification or spreading outside of the siRNA target site.

A genome-wide RNAi screen identifies RNAi and miRNA pathway components.
Sylvia E. J. Fischer, Qi Pan, Peter C. Breen, Taiowa A. Montgomery, Chi Zhang, Ellen Rim and Gary Ruvkun, data generation and analysis finished, writing paper.

We developed a fusion gene that enabled a full genome RNAi screen for enhanced siRNA or enhanced miRNA silencing. This fusion gene bears a dominant mutation in the collagen gene rol-6 that causes animals to move in an easily scorable rolling motion fused to the lin-14 3’ UTR which mediates the translational silencing of the lin-14 open reading frame or a lacZ reporter gene by the miRNAs lin-4 and let-7. The fusion gene was designed to study how mutations or gene inactivations that disable the miRNA pathway might fail to silence the fusion gene bearing the lin-14 3’ UTR. However, even though a similar lacZ fusion gene bearing the lin-14 3‘ UTR is silenced by lin-4 in wild type, the rol-6/lin-14 3’ UTR fusion gene is not repressed by lin-4 in wild type. The rol-6/lin-14 fusion gene is silenced by mutants that enhance RNAi and transgene silencing (for example the eri-1 mutant, or eri-6/7 RNAi, or synMuvB mutant RNAi) and non rolling animals are easily observed. This transgene was used in a full genome screen to discover gene inactivations that either enhance transgene silencing or enhance the ability of the miRNAs to repress via the lin-14 3’ UTR. Transgenic animals were exposed to dsRNA corresponding to 16,557 genes and scored for a rolling phenotype. After ruling out suppressors of the rol-6 phenotype, 69 gene inactivations cause silencing of the rol-6::lin-14 3’ UTR transgene. By testing all the hits from the primary screen with a panel of simpler transgenes without the lin-14 UTR, we classified the hits as enhancing RNAi or miRNAs. We detected dozens of gene inactivations that generally increase transgene silencing and RNAi, and a handful of gene inactivations that specifically enhance miRNA silencing. Available viable mutants corresponding to these genes were tested for enhanced RNAi and RNAi-defective phenotypes and many showed defects. Using additional molecular assays for siRNA silencing, identified new genes that act in ERGO-1 pathway: puf-8 RNA binding protein, F29A7.6/phosphoprotein, T26A8.4/CCCH Zn Finger protein, tcer-1/CA150 transcriptional elongation factor, ent-1/nucleoside transporter, mel-32/serine hydroxymethyltransferase which modifies uracil to thymidylate in the nucleus, C25F9.11/novel, B0001.6/novel, F57C9.7/novel, rde-4/RNA helicase and F58G11.2/RNA helicase. It also identified several factors in clathrin-dependent endocytosis that affect silencing. Non-lethal mutant alleles annotated to act in endocytosis showed an enhanced response to dsRNA: dpy-23/AP2 adaptin complex, chc-1/clathrin heavy chain, and rme-4/DENN domain. Our model for endocytosis involvement is that it may resolve a cycle of siRNA activity, so that in the absence of endocytosis, siRNAs remain active longer. These siRNAs could act at the primary step of production from dsRNA templates or at the amplification step by RdRps.

The gene inactivations that only enhanced silencing of a lin-14 3’UTR fusion gene identify negative regulators of microRNA activity. The vig-1/RNA binding protein, ccr-4/ CCR4-NOT transcription complex subunit 6, the P granule component exos-1/ exosomal 3’-5’ exoribonuclease ski4, htz-1/ H2A.Z histone variant, sams-3/4/ S-adenosylmethionine synthetase and F58G11.2/RNA helicase gene inactivations may enhance the silencing potency of miRNAs. Quantitative RT-PCR assays measuring lin-14 mRNA and mir-35 microRNA showed increased lin-14 mRNA levels in vig-1, ccr-4 and npp-12 mutant embryos, and reduced microRNA levels in vig-1, npp-12, and prmt-1 mutants and arl-8(RNAi).

mut-16 and other mutator-class genes modulate 22G and 26G siRNA pathways in Caenorhabditis elegans. (published Zhang. C., TA Montgomery, HW Gabel, SE Fischer, CM Phillips, N Fahlgren, C Sullivan, JC Carrington, and G. Ruvkun. 2011. mut-16 and other mutator-class genes modulate 22G and 26G siRNA pathways in Caenorhabditis elegans. Proc. Natl. Acad. Sci., 2011 108:1201-8.

Mutator-class (mut) genes mediate siRNA-guided repression of transposons as well as exogenous RNAi, but their roles in endogenous RNA silencing pathways are not well understood. To characterize the endogenous small RNAs dependent on mutator-class genes, small RNA populations from a null allele of mut-16 was subjected to deep sequencing. Additionally, each of the mutator-class genes was tested for a requirement in 26G siRNA pathways. The results indicate that mut-16 is an essential factor in multiple endogenous germline and somatic siRNA pathways involving several distinct Argonautes and RNA-dependent RNA polymerases. We also found that the soma-specific mut-16(mg461) mutant allele is present in multiple C. elegans laboratory strains, suggesting a possible advantage to this allele in the laboratory setting.

RDE-10/RDE-11 complex regulates RNAi and endogenous siRNA pathways in Caenorhabditis elegans. Current Biology, in press Zhang. C., TA Montgomery, SE Fischer, CM Phillips, N Fahlgren, C Sullivan, JC Carrington, and G. Ruvkun.

From a forward genetic screen for factors required for RNAi, we identified rde-10 and through proteomic analysis of RDE-10-interacting proteins, we identified a protein complex containing the new RNAi factor RDE-11, the RNAi factor RSD-2 as well as other candidate RNAi factors. The newly identified genes rde-10 and rde-11 encode a novel proteins. Mutations in rde-10 and rde-11 are resistant to low dosage, but sensitive to high dosage of double-stranded RNAs. Exogenous siRNA levels are greatly reduced in rde-10 and rde-11 mutants, indicating that they are required for siRNA formation or stability during RNAi. We assessed the roles of rde-10, rde-11, rsd-2 and rsd-6 in endogenous small RNA pathways using high-throughput RNA sequencing. These genes are required for a subset of secondary siRNAs derived from repeated C. elegans genes. Some of the RDE-10 interacting proteins have been identified as potential regulators of small RNA pathways in our previous RNAi-based genome-wide screens or proteomics; MATH-33 protein is a highly conserved ubiquitin carboxyl-terminal hydrolase and is required for cosuppression, an RNAi-related pathway, CAR-1 is an LSM RNA binding protein and PAB-1 is a multiple RRM poly A binding protein—both are components of the germline P granule, a site of small RNA activity.

Cytological analysis of the RNAi factors mut-2, mut-7, mut-16 and their roles in transposon silencing and RNAi. Carolyn Phillips, Genes and Development in review

In C. elegans, there are six families of transposons (Tc1-Tc5, Tc7). RNAi silences Tc1 in the germline: mutator (mut) class genes that activate transposition in the germline, also cause defects in RNAi. These genes includes a nucleotidyl transferase (mut-2), a 3’-5’ exonuclease (mut-7), a DEAD box RNA helicase (mut-14), a glutamine/asparagine rich protein (mut-16), two proteins of unknown function (rde-2 and mut-15). Functional GFP fusions to mut-7, rde-2, and mut-16 that rescue the corresponding mutants for temperature-sensitive sterility, production of endogenous siRNAs, and response to exogenous RNAi, are each visible throughout the germline and display distinct perinuclear localization. The pattern of localization is reminiscent of germline P-granules, which are nuclear pore-associated RNA-rich cytoplasmic germ granules. P-granules have previously been shown to contain numerous RNA-binding proteins including known components of small RNA pathways (2). MUT-16:GFP localizes to these foci in the absence of each of the other Mutator-class proteins, but the other GFP fusions fail to localize in a mut-16 mutant.

Identification of new small RNA pathway genes from correlated phylogenetic profiles and other genome analyses. Yuval Tabach, Nature submitted.

Hundreds of candidate protein cofactors for small RNA pathways have emerged from our functional genomic screens for defects in small RNA pathways. The Argonaute class proteins, which are central players in RNAi and miRNA, are entirely missing in 9 out of 31 species of Ascomycota, in 1 of 3 species of Basidomycota, in 6 of 14 protist species, and in one of two green algae species, but have not been lost in any of the 33 animal or 6 land plant species compared. The loss of the Argonaute correlates with reported competences to inactivate genes by RNA interference in those species that have been tested. To Identify other genes that share an evolutionary history with validated small RNA pathway proteins, we determined the phylogenetic profiles of all 20,000 proteins encoded by C. elegans genes in 85 disparate animal, fungal, plant and protist genomes. Genes that have coevolved but bear no homology to Argonautes were identified, for example the asparaginase/threonine aspartase/taspase encoded by K01G5.9, implicated in cleavage of the Mixed-Lineage Leukemia/trithorax histone methyltransferase MLL (3), the CAND-1 elongation factor and another elongation factor, THOC-1. THO complex members have emerged from genetic screens for defective transgene and RNAi silencing in Arabidopsis thaliana as well (4).
We used a full genome RNAi screen for gene inactivations that suppress the transgene silencing up-regulation of the eri-1 mutant as an experimental test of these phylogenetic profile predictions. C. elegans tandem array transgenes are silenced in the germline. Such transgenes are also silenced in many somatic cells in an eri-1 mutant. Screening animals grown on E coli strains expressing dsRNA targeting 18,578 genes, we identified 855 genes required for transgene silencing in the eri-1(mg366) background, including a number of previously identified genes such as rde-1 and dcr-1. The potency of transgene desilencing gene inactivations was ranked in four classes. The genes predicted to be RNAi cofactors from the phylogenetic profile analysis are highly enriched on these lists of candidate RNAi factors, including the strongest class of transgene desilencing gene inactivations.

*The Caenorhabditis elegans SOMI-1 zinc finger protein and SWI/SNF promote regulation of development by the mir-84 microRNA. Hayes, G. D. C.G Riedel, and G. Ruvkun. 2011. Genes Dev. 25:2079-92. *

The conserved miRNA let-7 and three paralogs of let-7, mir-84, mir-48 and mir-241, control the timing of stage-specific developmental events. These miRNAs are identical at the 5’ ends, but diverge at the 3’ ends. To identify factors required for the activity of mir-84 and possibly other miRNAs, we screened for mutations that suppress the developmental defects caused by overexpression of mir-84. Mutations in the somi-1 gene prevent these defects without affecting the expression level of mir-84. Loss of somi-1 also causes phenotypes similar to deletion of mir-84, showing that somi-1 is necessary for the normal function of this miRNA. somi-1 encodes a zinc-finger protein that localizes to nuclear foci and binds the promoters of let-60/RAS, lin-14 and lin-28, genes that may be targeted by mir-84 and similar miRNAs. Proteomic and genetic screens identified conserved chromatin-remodeling and homeodomain transcription factor complexes that work with somi-1 to regulate differentiation. Enrichment of SOMI-1 at the promoters of lin-14, lin-28 and let-60/RAS, transcripts of which are targets of let-7 and mir-84, and the requirement for somi-1 for the defects caused by misexpression of mir-84 and let-7, suggest that somi-1 mediates a nuclear response to the activity of these miRNAs.

The mevalonate pathway modulates microRNA activity via protein N-glycosylation. Zhen Shi, PNAS in press

A strong hit from our RNAi screen for miRNA defective gene inactivations encodes C. elegans HMG-CoA synthase, which synthesizes mevalonate, a key intermediate in the isoprenoid pathway, Inactivation of F25B4.6/HMG-CoA synthase by RNAi causes let-7 mutant-like phenotypes that are suppressed by the loss-of-function of validated let-7 target genes. The silencing of lin-14 mRNA translation by the lin-4 miRNA is also dependent on F25B4.6/HMG-CoA synthase: F25B4.6 RNAi causes a two fold derepression of LIN-14 protein levels. This de-silencing of LIN-14 by F25B4.6/HMG-CoA synthase inactivation is due to reduced lin-4 miRNA repression of lin-14 via its 3’ untranslated region. In the lin-14(n355gf) strain which lacks all the miRNA (lin-4 and let-7 and others) complementary sites in the lin-14 3’UTR, LIN-14 protein levels are not further increased when F25B4.6/HMG-CoA synthase is inhibited. The miRNA abundance is unaffected in F25B4.6/HMG-CoA synthase RNAi. These results show that F25B4.6/HMG-CoA synthase is required for the miRISC activity, downstream of miRNA biogenesis/accumulation.

Isoprenoids, the end products of the mevalonate pathway, feed into a wide range of downstream biosynthetic pathways in additional to the better known sterol synthesis: dolichol serves as the lipid carrier of the oligosaccharide moiety for protein N-glycosylation; ubiquinone and heme A function in the electron transport chain; prenylated proteins; and isopentenyl adenine, present in position 37 of tRNAs that read codons starting with U. Addition of mevalonate to plates rescues the let-7-deficient-like phenotypes of F25B4.6/HMG-CoA synthase inactivation, but not inactivation of the miRNA Argonaute alg-1/2 by RNAi. Cholesterol does not rescue the RNAi depletion of F25B4.6/HMG-CoA synthase. This indicates that instead of cholesterol, other biosynthetic product(s) of the mevalonate pathway modulate miRNA activity. HMG-CoA reductase (encoded by F08F8.2) also acts in the miRNA pathway. Homozygous F08F8.2(tm4368) animals arrest at the L1 stage. High concentration of mevalonate rescues the homozygous F08F8.2(tm4368) animals. F08F8.2(tm4368) homozygous mutant animals growing with low mevalonate fail to express the col-19::GFP in the let-7(mg279) mutant background. Statins inhibit HMG-CoA reductase activity. When fluvastatin is added to the growth medium, let-7(mg279 animals fail to express col-19::GFP, similar to the RNAi depletion of F25B4.6/HMG-CoA synthase or the F08F8.2(tm4368) mutation. Thus in C. elegans, the non-sterol output of the mevalonate pathway modulates miRNA activity.

Even though the RNA modification output was the most attractive model for how the mevalonate pathway intersects with miRNA regulation of target mRNAs, mass spectroscopy on nuclease P1 digested C. elegans 18-28nt small RNAs (~30% are miRNAs) did not detect N6-(∆2-isopentenyl)adenosine, whereas we could easily detect it in nuclease P1 treated C. elegans tRNA. We therefore broadened the search to include cherry-picked RNAi clones targeting the genes that function in protein prenylation, tRNA isopentenyltransferase, ubiquinone and heme A biosynthesis, dolichol synthesis and protein N-glycosylation. We performed an RNAi screen for gene inactivations that cause a col-19::GFP expression defect in the let-7(mg279) mutant background. The hits that cause failure of col-19::GFP expression all function in the pathway for protein N-glycosylation. The C. elegans oligosaccharyltransferase (OST) complex that carries out protein N-glycosylation has five subunits: the catalytic subunit T12A2.2/STT-3; and four accessory subunits: T22D1.4/ribophorin I, ostd-1, ostb-1 and dad-1. Knocking down any of these genes by RNAi causes col-19::GFP expression defect in the let-7(mg279) mutant, but not in wild type, suggesting a strong genetic interaction with let-7. Inactivation of the OST complex causes de-repression of miRNA targets, with no effect on the mature miRNA levels. This indicates that the activity, rather than the biogenesis or accumulation of miRNAs, requires the N-glycosylation.

Statins block cholesterogenesis by inhibiting HMG-CoA reductase, the rate-limiting enzyme in the mevalonate pathway and are prescribed to hundreds of millions of patients to lower the risk of heart diseases. While the efficacy of statins to lower cholesterol is the general view of their molecular mechanism, the central role that mevalonate regulation of N-glycosylation to in turn mediate the function of miRNA repression of target mRNAs suggest a miRNA axis of statin treatment of heart disease (5). Significantly, a deletion of the cardiac miRNA mir-208 strongly suppresses the high blood pressure induction of fibrosis caused by the adult stage misexpression of fetal myosin heavy chain gene (6). The mevalonate has also emerged from another miRNA analysis: mir-122 is required for the replication of hepatitus C virus and statins act in the same manner as loss of mir-122 to inhibit hepatitis C replication (7-9). mir-122 mediates the recruitment of HCV replication and associated small RNA proteins such as Argonautes to lipid vesicle replication zones within cells (8). The finding that in miRNA repression of target mRNAs in animal cells engages a vesicle sorting pathway is consistent with a sorting to such organelles in miRNA regulation of target mRNAs (10-12).

RNAs linked to the proteome. The small RNA field has now collected millions of small RNAs, via library construction and high throughput DNA sequencing approaches. We have been in the descriptive phase of small RNA biology for almost a decade: for the endogenous siRNAs, there are thousands of genes represented by antisense small RNAs and millions of distinct siRNAs that tile along mRNAs represented, but no clear function has emerged. Protein cofactors for small RNAs, identified either biochemically in assays for production of siRNAs or miRNAs or piRNAs, or genetically via screens for defective small RNA function have also emerged: For example, Dicer and the proteins of the Argonaute class that mediate the maturation and nuclease guidance activities and more recently, via RNAi screens for protein cofactors of small RNA pathways in our lab and other labs that revealed many other possible components that may sort and guide small RNAs to their targets (1,2).

We believe that the clever small RNA cloning approaches used by the field have systematically missed key small RNAs that are modified at their 5’ and 3’ ends to dramatically change the ability of these RNAs to interact with the proteome and other molecules in the cell. For example, the now established 5’ ligation of dephospho-coenzyme A to RNAs allows such small RNAs to form disulphides covalently with cysteine residues on proteins (or to glutamate or aspartate residues via thioester bonds), to essentially bar code those proteins with small RNA moieties that can base pair with RNA, DNA, or other RNA-tagged proteins. Such modified proteins could then be sorted via these tags, or asssemble onto other nucleic acid scaffolds, such as the kinetochore, or scaffolds that interact with nucleic acids, such as the centrosome or cytoskeleton.

Dephospho coenzyme A has a ligatable 3’ OH. Acetyl coenzymeA as well as the synthetase that generates it is localized to the nucleus for acetylation of histones and other transcription factors. S adenosyl methionine also bearing a 3’ hydroxyl that could ligate to small RNAs. SAM is the methyl donor in a large array of methylation reactions, including methylation of histones, a connection that is not uninteresting with regard to small RNA pathways. The linkage of S adenosyl methionine or S adenosyl homocysteine to the 5’ end of small RNAs could also allow these RNAs to accompany histone methylases to the nucleus and perhaps ligate small RNAs as they transfer a methyl group. NAD. Notice that the 3’ OH of the ATP moiety could be ligated to RNA and has been shown to be ligated (4). The ligation of NAD to the 5’ end of small RNAs would allow them to participate in adenylation reactions of glutamate, aspartate and arginine, leaving the small RNA tagging the target protein. Similarly, poly ADP ribosylation could multiply tag proteins with small RNAs. Interestingly, the poly ADP polymerases pme-1 and pme-2 are the closest of all C. elegans proteins in our phylogenetic profile to the argonautes suggesting that poly ADP modification is correlated with small RNA pathways in animals, plants, fungi and protists.

Support for the idea of ligation of coenzyme A to the 5’ end of small RNAs has emerged from multiple experiments as well. Based on the concept that dephosphocoenzyme A could ligate to RNAs and to then allow them to interact with the cysteine proteome, we searched for incorporation of 35S into RNA preparations from C. elegans and S. pombe. We saw incorporation of 35S in a molecule of about 20 to 25 nt only in C. elegans, that co purifies with RNA in phenol extractions in the trireagent or SDS proteinase K digestion isolation, ethanol precipitation, and binding to RNA purification resins. We could see the expected charged tRNAs for Met and Cys in both C. elegans and S. pombe. However the 35S RNA, which we have named suRNA is not sensitive to various RNAses, suggesting that either it is highly modified or not an RNA. One concern was that sulfated glycosaminoglycans might be 35S-labeled in these preparations, though we did not expect such a narrow size range for such polysaccharide polymers. Treatment with a bacterially derived heparinase degrades the 20 to 25 nt 35S fraction band. However, the annotation of commercially bought heparinase II, isolated from Flavobacteria, is not definitive at all. It is possible that these enzymes are actually involved in disruption of small RNA pathways, especially suRNAs and that they are promiscuous for other sulfated polymers.

Even more strongly supporting the model that modified nucleotides are ligated to small RNAs is mass spectroscopy of E. coli RNA by the Liu group, who reported that succinyl coenzyme A is ligated to RNAs isolated from E. coli and other bacteria (4). Their mass spec analysis revealed a number of mystery peaks that might correspond to other ligations to RNAs, and proved that NAD is added in an analogous manner (5). The focus of their work is to reveal other enzymatic capabilities of RNAs, with major implications for the capacities in the RNA world.

We expect to find that protein elements of the centrosome for example, are ligated to siRNAs corresponding to the centromere elements of C. elegans and other organisms to mediate the binding of particular proteins to these DNA regions. We expect to find that proteins that form multisubunit complexes may use complementary small RNAs to form and disassemble complexes. Ligation of small RNAs to proteins allows the addition of a handle that can be a bar code for assembly of protein complexes and for interaction of proteins with other nucleic acids. In the same way that PCR primers allow us to bar code and preferentially amplify segments in the lab, a small RNA tag on a protein allows that protein to be recognized by other proteins bearing the complementary RNA tag.

Lifespan and metabolism
During the last 15 years, the insulin/IGF-1/daf-2 signaling pathway has been shown to be the most potent regulator of lifespan in C. elegans. Signaling from DAF-2 is mediated through the AGE-1 phosphatidylinositol 3-kinase (PI3K), PDK-1, and AKT-1/2 kinases, to antagonize DAF-16, orthologous to human FoxO, a forkhead transcription factor (1-8). The function of this pathway in mediating metabolism and aging is conserved in C. elegans, Drosophila, and mammals. Because so much of the insulin signaling pathway is conserved, the new components we discover in C. elegans will have broad relevance to mammalian insulin signaling and longevity control.

Genetic analysis in C. elegans continues to identify components of the pathway that are likely to reveal human variation in insulin-like signaling, with medical significance for diabetes and the understanding of how insulin signaling and analogous hormonal pathways couple chronological age to many late onset diseases. Using RNAi to screen for defects in daf-2 pathway mediated longevity regulation, we identified a comprehensive genetic network necessary for the longevity response to low daf-2 insulin/IGF1 signaling (9). Similarly, our proteomic analysis of insulin signaling components has identified other new and unstudied candidate genes to act in insulin signaling. The use of RNAi screens and proteomics in C. elegans is opportune for two reasons: first, the tissues where insulin signaling is key for metabolic control has changed dramatically over the past decade. No longer is an exploration of insulin signaling only in the liver or muscle or even pancreas definitive. Neural and adipose centers of insulin signaling have emerged (7). We have identified new protein components of insulin signaling from whole animal extracts, and our RNAi screens are done in the whole animal, so insulin signaling across tissues is surveyed. This is unlike mammalian insulin signaling functional genomics which may assay for insulin responses in tissue culture, but not in the physiological context of a whole organism. Aging and diabetes may be more physiological and endocrine, not easily modeled in cell culture. In this way, the C. elegans insulin signaling genetic system is better model system for human aging and human diabetes than human cell culture.

Gene activities that mediate the longevity regulation by C. elegans insulin-like signaling. Samuelson, A. V. , Carr, C. E., and G. Ruvkun. 2007. Gene activities that mediate increased lifespan of C. elegans insulin-like signaling mutants. Genes and Development 21:2976-94.

Genetic and RNA interference screens for lifespan regulatory genes have revealed that the daf-2 insulin-like signaling pathway plays a major role in C. elegans metablism and longevity. This pathway converges on the DAF-16 transcription factor to regulate aging by controlling the expression of a large number of genes. We conducted a genome-wide RNA interference screen to identify genes necessary for daf-2 mutants to survive nearly twice as long as wild type, and identified approximately 200 gene inactivations that shorten daf-2 lifespan to near that of wild type but have much more minor impact when inactivated in wild type. Of the gene inactivations identified in our screen, most enriched are genes annotated to mediate vesicle sorting. For example, compared to loss of daf-16, inactivation of Y65B4A.3 caused the greatest suppression of daf-2 phenotypes. Y65B4A.3 is homologous to human charged multivesicular body protein 6 and the myristolyated subunit of yeast ESCRT-III, the endosomal sorting complex required for transport of transmembrane proteins into the multivesicular body pathway to the lysosomal/vacuolar lumen. Sixteen other endocytosis/vesicular trafficking related genes also suppressed daf-2. One of the responses to a decline in daf-2 is dramatic upregulation of the DAF-16 target gene sod-3, a manganese superoxide dismutase. Thirty-four gene inactivations suppressed the daf-2-dependent induction of sod-3 expression, including smk-1/ protein phosphatase regulatory subunit, mag-1/exon junction complex component, F28D1.9/fatty acid transporter, and cua-1/cation transporter.

Proteomic exploration of the insulin signaling pathway (not yet published)

In the absence of insulin-like signaling, DAF-16/FoxO binds to the promoters of numerous metabolic and stress response genes and can either activate or repress their transcription by RNA Pol II. Such transcriptional outputs cause enhanced stress resistance and a change to fat storage metabolism, and enhanced longevity. Using a proteomic approach, we have identified dozens of candidate protein interactors with insulin-signaling components. We constructed functional translational fusions to epitope tags for the transcription factor FOXO/DAF-16 and one of its activators, SMK-1. FOXO/DAF-16 mediates the transcriptional output of insulin/IGF signaling. SMK-1 is predicted to be a regulatory subunit of protein phosphatase 4 and has genetically been shown to promote DAF-16 activity. We integrated the fusion protein constructs into the C. elegans genome and to study DAF-16/FOXO in variable states of activation, we used three different strain backgrounds: wild type, daf-2(e1370) a strong kinase domain mutant with low insulin like signaling, and daf-18(mg198)/PTEN phosphatase null allele with constitutively high insulin like signaling. Epitope-tagged DAF-16/FOXO from each strain grown in large quantity (tens of liters of growth medium and tens of ml of packed worms) was purified by immunoprecipitation from each of these strains. Co-purifying potential physical interactors of the fusion proteins were identified by mass spectrometry (LC-MS/MS).

In order to determine the background of these purifications, parallel control purifications from worms lacking the fusion proteins were performed. Table 1 shows a list of the most abundant binding partners to FOXO/DAF-16 after common background proteins are subtracted from this list, yielding 156 specific binding partners to DAF-16/FOXO. Many of the FOXO/DAF-16 binding partners have chromatin or stress response related annotations. To test their function in daf-2 regulation of lifespan, we inactivated each corresponding gene by RNAi and surveyed phenotypes expected for either enhancement or loss of DAF-16/FOXO activity. We tested each gene inactivation in wild type animals for an extension of lifespan, enhanced resistance to heat or oxidative stress or inappropriate expression of the DAF-16/FOXO-activated gene sod-3. Conversely, we tested in daf-2/insulin receptor mutant animals for suppression of extended lifespan, suppression of resistance to heat or oxidative stress or an inability to overexpress sod-3. Of 89 binding partners tested, 65% showed significant lifespan or stress resistance phenotypes in at least one and 32% in multiple assays. The genes identified by this screen are highly enriched for acting in the longevity pathway: 18 % of the DAF-16/FoxO binding partners are required for lifespan extension in daf-2(e1370), while a genome-wide screen for the same phenotype had a discovery rate near 1% (9).

Proteomic abundance and functional data were combined to create a short-list of the most promising binding partners of DAF-16/FOXO. The short-list is enriched for subunits of the chromatin remodeling complex SWI/SNF, i.e. its core subunit SWSN-1 and regulatory subunit SWSN-3. A third subunit, SWSN-8, was also identified by the proteomics – although at very low abundance. Shown in Figure 1 is the strong suppression of daf-2 increased lifespan by gene inactivation of swsn-1, whereas this gene inactivation has very modest effects on wild type lifespan.

This phenotype was diminished in daf-16(mgDf47) mutant worms, suggesting that SWI/SNF functions in the same pathway as DAF-16/FOXO. sod-3 expression is induced by inactivation of insulin like signaling in the daf-2(e1370) mutant. Inactivation by RNAi of daf-16 or of the SWI/SNF subunits swsn-1, swsn-4 and to a lesser extend swsn-8 suppressed this induction. We confirmed a requirement for SWI/SNF in the regulation of DAF-16/FOXO target genes by monitoring the mRNA levels of endogenous target genes in wild type, daf-2(e1370), daf-2(e1370);daf-16(mgDf47), daf-2(e1370);swsn-1(os22), daf-2(e1370);swsn-3(tm3647), and daf-2(e1370);swsn-4(os13) worms. We found that these DAF-16/FOXO target genes required presence of functional SWI/SNF for their regulation. SMK-1 co-purified with several subunits of protein phosphatase 4, confirming its predicted role as a regulatory subunit to this enzyme.

Lifespan regulation by evolutionarily conserved genes essential for viability Curran, S. P. and G. Ruvkun. 2007. PloS Genetics 3(4):e56

We have discovered that other arrest points induced by gene inactivations of core cellular components such as the ribosome or mitochondrion or cytoskeleton may be as highly regulated and as key to lifespan regulation as the insulin regulated dauer arrest point. In common with the dauer arrest point, these other arrest points are induced by environmental inputs, for example starvation or natural products that target conserved cellular components such as the mitochondrion or the ribosome. Our analysis suggests that the integrity of these core cellular components are assessed, either in cells that tend to be the most exposed to the environment, or in all cells, and that a signaling pathway to endocrine control of development and reproduction may operate.

To reveal this essential pathway signaling system, we screened the 2700 gene inactivations that cause larval arrest or reproductive arrest in C. elegans for increased adult lifespan by initiating the gene knockdown once the animal had reached adulthood, thus bypassing any developmental abnormalities. We identified 64 genes that can extend lifespan when inactivated post-developmentally. More than 90% of the genes we identified were conserved from yeast to humans. Our yield of 64 gene inactivations out of 2700 tested (~2.4%) is a four-fold increased yield than the previous 89 gene inactivations out of 16,000 screened (~0.6%), and a higher proportion of the gene inactivations cause large increases in longevity.

To classify the pathways represented by these new genes, we performed secondary assays: DAF-16 localization, sod-3 expression, arrested larval survival, suppression of polyglutamine aggregation, and aberrant fat metabolism and clustered the genes by the phenotypes observed. Our analysis placed some of these longevity genes within the insulin-signaling pathway, while others were independent of this pathway.

Stress resistance may contribute directly to extended longevity, and is co-regulated with longevity. We have observed a panel of a dozen stress-responsive GFP fusions in strains experiencing gene inactivations corresponding to a library of ~200 lifespan-extending RNAi clones. We tested a heat induced GFP fusion, hsp-16::GFP, an ER stress induced gene, hsp-4::GFP, and two mitochondrial stress induced genes, hsp-6::GFP and gst-4::GFP. Most of the longevity inducing gene inactivations activated one or more stress reporter genes and the pattern of stress reporter activation was characteristic for ribomsomal inactivation for example compared to mitochondrial inactivation.

The integrity of these core cellular components may be assessed in cells that tend to be the most exposed to the environment, and a signaling pathway to endocrine control of development and reproduction may operate. For example, we have found that there are sentinel signaling cells: the intestine where microbes are first encountered in an intimate way and sensory neurons that might “probe” the antibiotic environment before the rest of the cells of the animal are inhibited. We used a collection of strains in which gene knockdowns can be accomplished within a single, restricted tissue using the same technique of feeding animals bacteria expressing dsRNA against target genes. The strain backgrounds used for these experiments are fully defective for RNAi because they lack the necessary gene (either rde-1 or sid-1). Only the tissue(s) in which gene function is inactivated by RNAi of essential genes are those in which the tissue-specific promoters rescue rde-1 or sid-1 expression is rescued, in neurons or intestine or muscle. In addition, we used a mutant strain proficient for RNAi in the germline but not in somatic tissue (rrf-1) due to differing mechanisms of RNAi in these tissue types. Inactivation of core cell components in each tissue tested – the hypodermis, the intestine, the gonad and body wall muscle – is competent to trigger similar responses to essential gene inactivation as inactivation of these genes in all tissues. This suggests the existence of an endocrine system for response to essential gene inactivation. Similar endocrine outputs from mitochondrial gene inactivation have been found by the Dillin lab (12).

The developmental arrests induced by these gene inactivations and the longevity induction may be a “programmed” response to a deficiency in a key function, and active signaling programs may mediate the arrest point, as a sort of “developmental checkpoint”. Such an arrest program interpretation depends on two key attributes: 1. The arrest should be reversible. We have observed that when drug is removed in many cases, the arrest is reversible. 2. There should be mutations that disable the arrest program. This is also established below.

Stress decoupled mutants shorten the lifespan of insulin signaling and other long lived mutants (not published)

A common theme to many of the cellular components that induce increased longevity when inactivated is that many are targets of antibiotics produced by a range of fungi and microbes that nematodes encounter in the environment. We hypothesize that as a larvae or adult enters an environment with an antibiotic, there may be signaling pathways that detect, for example, antibiotic-induced ribosomal deficiency to trigger cessation of reproductive developmental trajectory, arrest at a particular developmental point, as well as longevity enhancing pathways and behavioral aversion programs to allow the arrested animal to escape and survive long enough to reanimate reproduction and be alive for that joyous experience. The induced stress adaptation and survival pathways would ensure that the animal could escape the antibiotic and resume reproductive development in a less toxic environment or feeding on less toxic microbes. Inhibition of translation by RNAi of translation factors may mimic the ribosomal function deficiency induced by antibiotics in the normal C. elegans ecosystem, and trigger the physiological response of developmental arrest and cessation of reproduction and initiation of longevity programs in the arrested larvae or adults. Exposure of C. elegans to many of these drugs cause similar developmental arrest and a modest increase in lifespan, but the drugs are not as potent as the gene inactivation by RNAi. This may be because drug detoxification is highly evolved —-they induce a variety of detoxification pathways that may be more effective on the small molecule drugs than on gene inactivation by RNAi.

The same stress reporter genes activated by the longevity inducing gene inactivations are also induced by the drugs. Tunicamycin is a natural product the bacterium Streptomyces Iysosuperficus; it inhibits ER N-linked glycosylation and strongly induces HSP-4, a component of the ER unfolded protein response. Antimycin is a natural product Streptomyces and inhibits the mitochondrial electon transport complex 3 by binding the cytochrome b subunit and induces hsp-6, a component of the mitochondrial unfold protein response.

We screened for new gene inactivations that cause a failure to induce the ER or mitochondrial or other stress GFP fusion genes under drug or stressed conditions—-these gene inactivations may cause animals to be “blind” to mitochondrial or ribosomal dysfunction, and therefore not induce these GFP fusion genes. We then asked if a failure to induce these genes reflects a general decoupling of the surveillance of the ribosome for example to the induction of longevity normally induced by essential gene inactivations. In this way, we will discern the signaling pathway, both within cells and between cells for decrements in core cellular functions.

We first did pilot screens starting with cherry picked sublibraries, and propose to ramp up to full genome screens in this proposal. Our first candidate genes for signaling from essential gene inactivation to xenobiotic resistance emerged from a survey of gene inactivations from four candidate sublibraries of 100 to 300 genes each:

1. gene inactivations that interfere with the longevity increase induced by low insulin signaling might also act in essential gene induction of longevity. So we screened a cherry picked library of gene inactivations that shorten the lifespan of daf-2 insulin like signaling mutants (9).
2. translational control of drug detoxification mechanisms is a likely mechanism because of the speed of response possible so that animals with defects in miRNA regulation of translation might be defective in their normal response to these drugs or mutations that affect the ribosome for example. Our lab has generated a set of 200 gene inactivations that disable let-7 miRNA regulation of its target mRNA lin-41. Many of these gene inactivations disable a broader range of miRNAs and we screened these 200 candidate gene inactivations for defects in drug induction of the GFP fusion genes (13).
3. because many of the C. elegans homologues of drug detoxification enzymes (cytochrome p450 and UDP glycosyltransferases and glutathione S transferases) are clustered in tandem arrays in the genome (14), we reasoned that genes identified to disable tandem array genetic elements might disable or inappropriately “preinduce” this xenobiotic response. Our lab has generated a set of 300 gene inactivations that disable or enable tandem array expression which we marshaled to this screen (15).

So far from these pilot screens, we have identified 30 gene inactivations that fail to activate the drug induced reporter genes as shown in Table 2 below. These genes come from all three candidate libraries. For example, inactivation of the genes ima-3, let-70 and the known gene ire-1 interfere with induction of hsp-4::GFP by tunicamycin. Another potent poison blind gene inactivation is pas-3, a proteasomal component, which is strongly defective in miRNA regulation by let-7 and lin-4. This gene inactivation is partially disabled in the mitochondrial drug induction of hsp-6. The genes identified by these inactivations are candidates for the genetic pathways that transduce deficiencies in ribosomal or mitochondrial function to endocrine pathways for developmental arrest and longevity induction.

If the cytoprotective pathways used as stress reporters in the studies above are normally induced by mutants or gene inactivations that confer increased longevity and are part of the program for increased longevity, a decoupling of their induction might shorten the lifespan of long lived mutants more than wild type. We tested whether the 30 gene inactivations with defects in cytoprotective gene induction also abrogated the increase in lifespan induced by mitochondrial dysfunction, reduced feeding, or disruption of insulin signaling. In these experiments, 12 of 30 gene inactivations tested abrogate 2/3 or more of the lifespan extension observed in eat-2, isp-1 and/or daf-2 mutants. While dcp-66, pas-3 and arf-3 exert their largest suppression of lifespan in isp-1, inactivation of cpf-2, wnk-1 and nekl-2 are most potent in the eat-2 mutant. phi-50, ima-3, gob-1, ufd-1, let-70, and elt-2 are critical to lifespan extension in both the isp-1 and eat-2 mutants. Two of these, phi-50 and ima-3, also reduce the lifespan of daf-2 mutants by more than 2/3. These gene inactivations are analogous to the ceh-23 suppression of mitochondrial mutant lifespan increase (16).

A soma-to-germline transformation phenotype in long-lived C. elegans mutants (Curran et al, Nature 459: 1079-84)

Genomic instability of somatic cells is a hallmark of increased age among most organisms. Protection of the germline on the other hand is an evolutionarily conserved trait. We uncovered a soma-to-germline transfomation phenotype among Caenorhabditis elegans longevity mutants. Mutations that inhibit insulin-like signaling cause misexpression of a germline restricted gene, pgl-1::gfp in the intestine and hypodermis. Mutants in the synMuvB class of genes are Eri and display somatic misexpression of the normally germline-limited P-granule component PGL-1. We monitored a PGL-1::GFP protein fusion in daf-2 or age-1 mutant strains. pie-1 and pgl-1 are exclusively expressed in the germline of wild type animals. Decreased insulin-signaling caused strong misexpression of PGL-1:GFP in hypodermal and intestinal somatic tissues of dauers and late larval stage animals. By qPCR, we could verify strong up-regulation of the P granule components pgl-1, pgl-2, and pgl-3 in the soma of daf-2 and age-1 mutants. This misexpression of germline components was strongly suppressed by mutations in the FoxO transcription factor DAF-16, the transcriptional output that is repressed by the insulin-like signaling pathway.

The misexpression of germline markers suggests that the somatic cells of an insulin-signaling mutant are more germline-like. Germline-transformed somatic cells, like germ cells, may engage additional protective pathways that prevent or slow genomic destabilization; the effect of which could cause an increased ability to respond to stress and extend lifespan. We tested the ability of daf-2 mutants to protect somatic tissues from genomic instability by feeding a sublibrary of RNAi clones previously shown to induced somatic nuclear DNA mutation. RNAi targeting rpa-2, srxa-6/dnJ-25, and F49E12.6 causes an increased rate of somatic mutation. These gene inactivations cause nuclear DNA damage as measured by the somatic expression of a normally out-of-frame lacZ transgene. Expression is induced following a nucleotide insertion/deletion event that places the lacZ gene in-frame. After feeding these RNAi clones, X-gal staining of animals carrying this transgene in the daf-2(e1368) background displayed reduced lacZ expression in somatic cells compared to animals harboring the transgene alone, suggesting a reduction in genomic instability in the somatic cells of the insulin-like signaling mutants.

If the misexpression of germline genes in somatic tissues contributes to the longevity phenotype of insulin signaling mutants then removal of the genes by RNAi should suppress the longevity phenotype. To test this we used RNAi to ablate the misexpressed germline transcripts in daf-2 mutant animals. Depletion of these misexpressed germline genes in the daf-2 mutant reduced the lifespan of these normally long-lived animals but not wild type animals. The enhanced RNAi phenotype of synMuvB mutations is suppressed by loss of function mutations in chromatin remodeling ATPase, isw-1 and the SET domain containing protein, mes-4. Similarly, RNAi towards isw-1 and mes-4 could partially suppress the enhanced longevity of daf-2(e1368).

We tested whether any of the other genes identified in our post-developmental RNAi screen to extend lifespan also displayed somatic cell to germline cell fate transformations by tracking the expression of endogenous PGL-1. PGL-1 is normally restricted to the germline where it forms perinuclear punctate structures around mitotic and meiotic germcells. RNAi clones targeting two components of the cytosolic chaperonin complex cct-4 and cct-6 also caused somatic expression of PGL-1. The somatic PGL-1 protein localized to punctate perinuclear rings in hypodermal cells and also produced intestinal cytoplasmic granules.

Germ cells employ protective mechanisms to ensure genetic integrity. The soma-to-germline transformation provides a potential mechanism for the observation that long-lived mutants exhibit resistance to genotoxic stress. We hypothesize that increased genomic stability in somatic cells may be an important pathway facilitating lifespan extension. It will be interesting to investigate if this is a broadly conserved mechanism given that protection of the germline is a shared trait across species.

We have discovered that the daf-2 insulin/IGF1 pathway also regulates the intensity of RNAi in C. elegans. Long-lived mutants in daf-2 and age-1 are enhanced for RNAi and misexpress the normally germline restricted gene P granule RNAi components in the intestine and hypodermis 109-111 112, 113. Mutants in the synMuvB class of genes are also enhanced for RNAi (Eri) and display somatic misexpression of the normally germline-limited P-granule components. The misexpression of germline markers suggests that the somatic cells of an insulin-signaling mutant are more germline-like. Germline-transformed somatic cells, like germ cells, engage additional protective pathways that prevent or slow genomic destabilization to cause an increased ability to respond to stress and extend lifespan. The germline RNAi components that we study are universal to animals. The genes we will discover in this proposal that mediate the regulation of the P granule components are likely to be universal to all animals and to act in aging pathways of humans as well.

Our genetic analysis of aging in the reproductive system of C. elegans is thematically related to the regulation of germ cell pathways in somatic cells. We have found many gene inactivations affect reproductive senescence, some of which also affect organismal aging, some of which do not. Because the reproductive system in mammals is tightly coupled to an endocrine system that assesses nutritional and stress status, our genetic analysis of this system in C. elegans is likely to reveal previously unknown endocrine axes and signals.

Regulation of RNAi by the daf-2 insulin-like signaling pathway (published as Wang, D. and G. Ruvkun. 2004. The insulin pathway regulates RNAi in C. elegans. Cold Spring Harbor Symposium on Quantitative Biology, 69th edition, 69: 429-433.).

C.elegans strains with decreased insulin-like signaling have a more intense RNAi response tha

Tiny RNA Biology

Small RNA pathways regulate gene expression, chromatin structure and defense against invading elements such as transposons and viruses. Small RNA deep sequencing experiments have shown that about half of all genes produce endogenous siRNAs suggesting that this regulatory axis controls a wide range of gene activities. siRNAs are incorporated into effector complexes, comprised of an Argonaute protein and accessory factors, which direct silencing of complementary RNAs and in certain species, such as C. elegans, recruit RNA-dependent RNA polymerases (RdRPs) to the target, resulting in siRNA amplification. All small RNAs act sequence-specifically through base pairing with their target mRNA, but the outcome of the small RNA:target interaction can vary from suppression of transcription to mRNA degradation or translational repression and this is likely governed in part by the specific Argonaute cofactor and in part by other coupled proteins that mediate the sorting of these small RNAs within the cell and between cells. Much remains to be learned about miRNAs and siRNAs and how they silence gene expression. There are hints that they sequester target mRNAs in P bodies and other subcellular structures, and constitute elements in an RNA trafficking system. There are hints of extracellular movement of small RNAs, perhaps constituting a shadow endocrine system. The genes identified are likely to be key factors in the function of small RNAs and their identification may enable more potent RNAi based drug development.

Our work follows up on our past full genome screens for candidate cofactor genes and our development of molecular signatures for small RNA defects using deep sequencing. The gene lists identify the key elements in the choreography of small RNAs in C. elegans. Because the genes are nearly all conserved across phylogeny, it is likely that the rules we work out in C. elegans will apply generally. Our goals are to use our deep sequencing analyses and new reporter genes for endogenous siRNAs and miRNAs to transform these lists of about 500 genes into coherent pathways of RNA processing, trafficking and presentation to the genome. We expect that the implications of the work will be to discover to new elements in how miRNAs and siRNAs are generated and engage cellular proteins to in turn engage their targets, as well as the discovery of other small RNA pathways from our analysis of the genes we have identified.

There have been 2 general areas of small RNA biology that we have developed: the mechanisms of exogenous and endogenous RNAi and the mechanism of miRNA regulation of target mRNAs.

The exonuclease ERI-1 plays a conserved dual role in RNA interference and ribosomal 5.8S RNA processing. Gabel, H. W. and G. Ruvkun. 2008. Nat Struct Mol Biol. 15: 531-3.

In C. elegans and fungi and plants, RNA dependent RNA polymerases amplify primary siRNAs to produce a more robust response. This two stage amplifier with feedforward amplification, like PCR, can amplify noise, so as in good electronic design, is likely to be attenuated. To identify negative regulators of C. elegans RNAi, we carried out a genetic screen for mutants that show an enhanced RNAi (Eri) phenotype. Mutants in eri-1 display an enhanced RNA interference (Eri) phenotype in which dsRNAs that are ineffective in silencing target mRNAs in wild-type trigger robust silencing in an eri-1 null mutant. eri-1 has a conserved endogenous function that was previously unsuspected: the 5.8s RNA inappropriately retains 1 to 4 nucleotides of its precursor in the eri-1 mutant compared to wild type. The homolog of ERI-1 in the fission yeast S. pombe also negatively regulates RNAi and we also observed that the 5.8S rRNA in the S. pombe eri1∆ mutant has 2 to 8 additional 3’ nucleotides. The Rao group at Harvard also discovered that mammalian ERI-1 targets the 5.8S RNA analogously (1). Thus ERI-1 plays a conserved dual role in the RNAi and rRNA processing pathways.

Trans-splicing in C. elegans generates the negative RNAi regulator ERI-6/7. Fischer, S.E.J., M. D. Butler, Q. Pan and G. Ruvkun 2008, Nature 455: 491-6.

Two other mutants from the enhanced RNAi genetic screen, eri-6 and eri-7 map to two adjacent ORFs present on opposite strands but failed to complement. The syntenic region in C. briggsae revealed that eri-6 and eri-7 constitute one gene on one strand in this species. The eri-6/7 protein is a member of the superfamily I DNA and RNA helicases. The divergently transcribed RNAs for eri-6 and eri-7 are assembled in C. elegans into one mRNA by trans-splicing. A 930 bp repeat 3’ to eri-6 and 5’ to eri-7 mediates this trans-splicing. We proved this by the detection adenosine to inosine editing of the proposed RNA duplex mediated by an adenosine deaminase (ADAR). A strain with mutations in both adenosine deaminase genes enhances transgene silencing, suggesting that editing of dsRNAs normally inhibits RNAi. One model for the increase in transgene silencing the absence of these ADARs is that the dsRNA element in eri-6/7 is a better target for the RNAi pathway when it is not edited, thus decreasing eri-6/7 gene activity.

The ERI-6/7 Helicase Acts at the First Stage of an siRNA Amplification Pathway that Targets Recent Gene Duplications (Fischer, SEJ., TA Montgomery, C Zhang, N Fahlgren, A Hwang, CM Sullivan, JC Carrington and G Ruvkun 2011, PloS Genetics 7(11): e1002369.

To characterize the role of eri-6/7 in endogenous siRNA pathways, we compared the small RNA profiles of eri-6/7 mutants as well as ergo-1 and eri-1 mutants to wild type. siRNAs corresponding to about 80 target genes were depleted in eri-6/7 mutants and the corresponding mRNA levels were up-regulated. The genes targeted by eri-6/7 show extensive nucleotide homology to each other but are not conserved between C. elegans and C. briggsae. These genes also have few introns, supporting the model that these genes have recently been acquired by C. elegans, perhaps from viruses. ERGO-1/ERI-6/7 pathway may constitute elements of such a viral surveillance pathway that extends beyond the initial infection.

An endogenous siRNA sensor reporter gene to easily assay the production or response to endogenous C. elegans siRNAs Montgomery TA, E. Rim, N Fahlgren, C Sullivan, JC Carrington, and G. Ruvkun.).

We developed a GFP based sensor for endogenous siRNA activity in C. elegans. 22G siR-1 is derived from a cluster of 22G siRNAs on the X chromosome that are dependent on embryonically expressed upstream ERGO-1 generated 26G siRNAs. The siRNA sensor, ubl-1::gfp-siR-1-sensor, contains a single engineered perfect duplex target site for an abundant endogenous siRNA, 22G siR-1, embedded in the 3’ UTR of ubiquitin-like1 (ubl-1) and expressed under the control of the ubl-1 promoter, which is active in all tissues throughout development (Figure 1). A control construct ubl-1::gfp, lacks the siRNA target site. Each construct was introduced into C. elegans using Mos1-mediated single copy insertion. GFP expression was ubiquitous in C. elegans containing the control, which lacks the 22G siR-1 target site, but was nearly absent in C. elegans containing the reporter with the 22G siR-1 sensor element in the 3’ UTR. The silencing of the siRNA sensor is suppressed by ergo-1 or eri-6/7 RNAi.

To determine if siRNAs trigger production of siRNAs in the genomic vicinity of the initial target site, we deep sequenced small RNAs from C. elegans containing either the ubl-1::gfp or ubl-1::gfp-siR-1-sensor transgene. 22G siRNAs were uniformly distributed across both transgenes and were derived exclusively from coding and vector sequence and not from the ubl-1 5’ and 3’ untranslated regions (Figure 1CD). A large peak was observed at the siRNA target site of the sensor, it is 22G siR-1 that may originate from the endogenous X-cluster siRNA locus, as the levels of 22G siR-1 were identical between control- and siRNA sensor-transgenic C. elegans. But this abundant siRNA does not attract more siRNAs to the region. These results suggest that, unlike primary exogenous siRNAs and endogenous 26G siRNAs, 22G siRNAs do not trigger siRNA amplification or spreading outside of the siRNA target site.

A genome-wide RNAi screen identifies RNAi and miRNA pathway components.
Sylvia E. J. Fischer, Qi Pan, Peter C. Breen, Taiowa A. Montgomery, Chi Zhang, Ellen Rim and Gary Ruvkun, data generation and analysis finished, writing paper.

We developed a fusion gene that enabled a full genome RNAi screen for enhanced siRNA or enhanced miRNA silencing. This fusion gene bears a dominant mutation in the collagen gene rol-6 that causes animals to move in an easily scorable rolling motion fused to the lin-14 3’ UTR which mediates the translational silencing of the lin-14 open reading frame or a lacZ reporter gene by the miRNAs lin-4 and let-7. The fusion gene was designed to study how mutations or gene inactivations that disable the miRNA pathway might fail to silence the fusion gene bearing the lin-14 3’ UTR. However, even though a similar lacZ fusion gene bearing the lin-14 3‘ UTR is silenced by lin-4 in wild type, the rol-6/lin-14 3’ UTR fusion gene is not repressed by lin-4 in wild type. The rol-6/lin-14 fusion gene is silenced by mutants that enhance RNAi and transgene silencing (for example the eri-1 mutant, or eri-6/7 RNAi, or synMuvB mutant RNAi) and non rolling animals are easily observed. This transgene was used in a full genome screen to discover gene inactivations that either enhance transgene silencing or enhance the ability of the miRNAs to repress via the lin-14 3’ UTR. Transgenic animals were exposed to dsRNA corresponding to 16,557 genes and scored for a rolling phenotype. After ruling out suppressors of the rol-6 phenotype, 69 gene inactivations cause silencing of the rol-6::lin-14 3’ UTR transgene. By testing all the hits from the primary screen with a panel of simpler transgenes without the lin-14 UTR, we classified the hits as enhancing RNAi or miRNAs. We detected dozens of gene inactivations that generally increase transgene silencing and RNAi, and a handful of gene inactivations that specifically enhance miRNA silencing. Available viable mutants corresponding to these genes were tested for enhanced RNAi and RNAi-defective phenotypes and many showed defects. Using additional molecular assays for siRNA silencing, identified new genes that act in ERGO-1 pathway: puf-8 RNA binding protein, F29A7.6/phosphoprotein, T26A8.4/CCCH Zn Finger protein, tcer-1/CA150 transcriptional elongation factor, ent-1/nucleoside transporter, mel-32/serine hydroxymethyltransferase which modifies uracil to thymidylate in the nucleus, C25F9.11/novel, B0001.6/novel, F57C9.7/novel, rde-4/RNA helicase and F58G11.2/RNA helicase. It also identified several factors in clathrin-dependent endocytosis that affect silencing. Non-lethal mutant alleles annotated to act in endocytosis showed an enhanced response to dsRNA: dpy-23/AP2 adaptin complex, chc-1/clathrin heavy chain, and rme-4/DENN domain. Our model for endocytosis involvement is that it may resolve a cycle of siRNA activity, so that in the absence of endocytosis, siRNAs remain active longer. These siRNAs could act at the primary step of production from dsRNA templates or at the amplification step by RdRps.

The gene inactivations that only enhanced silencing of a lin-14 3’UTR fusion gene identify negative regulators of microRNA activity. The vig-1/RNA binding protein, ccr-4/ CCR4-NOT transcription complex subunit 6, the P granule component exos-1/ exosomal 3’-5’ exoribonuclease ski4, htz-1/ H2A.Z histone variant, sams-3/4/ S-adenosylmethionine synthetase and F58G11.2/RNA helicase gene inactivations may enhance the silencing potency of miRNAs. Quantitative RT-PCR assays measuring lin-14 mRNA and mir-35 microRNA showed increased lin-14 mRNA levels in vig-1, ccr-4 and npp-12 mutant embryos, and reduced microRNA levels in vig-1, npp-12, and prmt-1 mutants and arl-8(RNAi).

mut-16 and other mutator-class genes modulate 22G and 26G siRNA pathways in Caenorhabditis elegans. (published Zhang. C., TA Montgomery, HW Gabel, SE Fischer, CM Phillips, N Fahlgren, C Sullivan, JC Carrington, and G. Ruvkun. 2011. mut-16 and other mutator-class genes modulate 22G and 26G siRNA pathways in Caenorhabditis elegans. Proc. Natl. Acad. Sci., 2011 108:1201-8.

Mutator-class (mut) genes mediate siRNA-guided repression of transposons as well as exogenous RNAi, but their roles in endogenous RNA silencing pathways are not well understood. To characterize the endogenous small RNAs dependent on mutator-class genes, small RNA populations from a null allele of mut-16 was subjected to deep sequencing. Additionally, each of the mutator-class genes was tested for a requirement in 26G siRNA pathways. The results indicate that mut-16 is an essential factor in multiple endogenous germline and somatic siRNA pathways involving several distinct Argonautes and RNA-dependent RNA polymerases. We also found that the soma-specific mut-16(mg461) mutant allele is present in multiple C. elegans laboratory strains, suggesting a possible advantage to this allele in the laboratory setting.

RDE-10/RDE-11 complex regulates RNAi and endogenous siRNA pathways in Caenorhabditis elegans. Current Biology, in press Zhang. C., TA Montgomery, SE Fischer, CM Phillips, N Fahlgren, C Sullivan, JC Carrington, and G. Ruvkun.

From a forward genetic screen for factors required for RNAi, we identified rde-10 and through proteomic analysis of RDE-10-interacting proteins, we identified a protein complex containing the new RNAi factor RDE-11, the RNAi factor RSD-2 as well as other candidate RNAi factors. The newly identified genes rde-10 and rde-11 encode a novel proteins. Mutations in rde-10 and rde-11 are resistant to low dosage, but sensitive to high dosage of double-stranded RNAs. Exogenous siRNA levels are greatly reduced in rde-10 and rde-11 mutants, indicating that they are required for siRNA formation or stability during RNAi. We assessed the roles of rde-10, rde-11, rsd-2 and rsd-6 in endogenous small RNA pathways using high-throughput RNA sequencing. These genes are required for a subset of secondary siRNAs derived from repeated C. elegans genes. Some of the RDE-10 interacting proteins have been identified as potential regulators of small RNA pathways in our previous RNAi-based genome-wide screens or proteomics; MATH-33 protein is a highly conserved ubiquitin carboxyl-terminal hydrolase and is required for cosuppression, an RNAi-related pathway, CAR-1 is an LSM RNA binding protein and PAB-1 is a multiple RRM poly A binding protein—both are components of the germline P granule, a site of small RNA activity.

Cytological analysis of the RNAi factors mut-2, mut-7, mut-16 and their roles in transposon silencing and RNAi. Carolyn Phillips, Genes and Development in review

In C. elegans, there are six families of transposons (Tc1-Tc5, Tc7). RNAi silences Tc1 in the germline: mutator (mut) class genes that activate transposition in the germline, also cause defects in RNAi. These genes includes a nucleotidyl transferase (mut-2), a 3’-5’ exonuclease (mut-7), a DEAD box RNA helicase (mut-14), a glutamine/asparagine rich protein (mut-16), two proteins of unknown function (rde-2 and mut-15). Functional GFP fusions to mut-7, rde-2, and mut-16 that rescue the corresponding mutants for temperature-sensitive sterility, production of endogenous siRNAs, and response to exogenous RNAi, are each visible throughout the germline and display distinct perinuclear localization. The pattern of localization is reminiscent of germline P-granules, which are nuclear pore-associated RNA-rich cytoplasmic germ granules. P-granules have previously been shown to contain numerous RNA-binding proteins including known components of small RNA pathways (2). MUT-16:GFP localizes to these foci in the absence of each of the other Mutator-class proteins, but the other GFP fusions fail to localize in a mut-16 mutant.

Identification of new small RNA pathway genes from correlated phylogenetic profiles and other genome analyses. Yuval Tabach, Nature submitted.

Hundreds of candidate protein cofactors for small RNA pathways have emerged from our functional genomic screens for defects in small RNA pathways. The Argonaute class proteins, which are central players in RNAi and miRNA, are entirely missing in 9 out of 31 species of Ascomycota, in 1 of 3 species of Basidomycota, in 6 of 14 protist species, and in one of two green algae species, but have not been lost in any of the 33 animal or 6 land plant species compared. The loss of the Argonaute correlates with reported competences to inactivate genes by RNA interference in those species that have been tested. To Identify other genes that share an evolutionary history with validated small RNA pathway proteins, we determined the phylogenetic profiles of all 20,000 proteins encoded by C. elegans genes in 85 disparate animal, fungal, plant and protist genomes. Genes that have coevolved but bear no homology to Argonautes were identified, for example the asparaginase/threonine aspartase/taspase encoded by K01G5.9, implicated in cleavage of the Mixed-Lineage Leukemia/trithorax histone methyltransferase MLL (3), the CAND-1 elongation factor and another elongation factor, THOC-1. THO complex members have emerged from genetic screens for defective transgene and RNAi silencing in Arabidopsis thaliana as well (4).
We used a full genome RNAi screen for gene inactivations that suppress the transgene silencing up-regulation of the eri-1 mutant as an experimental test of these phylogenetic profile predictions. C. elegans tandem array transgenes are silenced in the germline. Such transgenes are also silenced in many somatic cells in an eri-1 mutant. Screening animals grown on E coli strains expressing dsRNA targeting 18,578 genes, we identified 855 genes required for transgene silencing in the eri-1(mg366) background, including a number of previously identified genes such as rde-1 and dcr-1. The potency of transgene desilencing gene inactivations was ranked in four classes. The genes predicted to be RNAi cofactors from the phylogenetic profile analysis are highly enriched on these lists of candidate RNAi factors, including the strongest class of transgene desilencing gene inactivations.

*The Caenorhabditis elegans SOMI-1 zinc finger protein and SWI/SNF promote regulation of development by the mir-84 microRNA. Hayes, G. D. C.G Riedel, and G. Ruvkun. 2011. Genes Dev. 25:2079-92. *

The conserved miRNA let-7 and three paralogs of let-7, mir-84, mir-48 and mir-241, control the timing of stage-specific developmental events. These miRNAs are identical at the 5’ ends, but diverge at the 3’ ends. To identify factors required for the activity of mir-84 and possibly other miRNAs, we screened for mutations that suppress the developmental defects caused by overexpression of mir-84. Mutations in the somi-1 gene prevent these defects without affecting the expression level of mir-84. Loss of somi-1 also causes phenotypes similar to deletion of mir-84, showing that somi-1 is necessary for the normal function of this miRNA. somi-1 encodes a zinc-finger protein that localizes to nuclear foci and binds the promoters of let-60/RAS, lin-14 and lin-28, genes that may be targeted by mir-84 and similar miRNAs. Proteomic and genetic screens identified conserved chromatin-remodeling and homeodomain transcription factor complexes that work with somi-1 to regulate differentiation. Enrichment of SOMI-1 at the promoters of lin-14, lin-28 and let-60/RAS, transcripts of which are targets of let-7 and mir-84, and the requirement for somi-1 for the defects caused by misexpression of mir-84 and let-7, suggest that somi-1 mediates a nuclear response to the activity of these miRNAs.

The mevalonate pathway modulates microRNA activity via protein N-glycosylation. Zhen Shi, PNAS in press

A strong hit from our RNAi screen for miRNA defective gene inactivations encodes C. elegans HMG-CoA synthase, which synthesizes mevalonate, a key intermediate in the isoprenoid pathway, Inactivation of F25B4.6/HMG-CoA synthase by RNAi causes let-7 mutant-like phenotypes that are suppressed by the loss-of-function of validated let-7 target genes. The silencing of lin-14 mRNA translation by the lin-4 miRNA is also dependent on F25B4.6/HMG-CoA synthase: F25B4.6 RNAi causes a two fold derepression of LIN-14 protein levels. This de-silencing of LIN-14 by F25B4.6/HMG-CoA synthase inactivation is due to reduced lin-4 miRNA repression of lin-14 via its 3’ untranslated region. In the lin-14(n355gf) strain which lacks all the miRNA (lin-4 and let-7 and others) complementary sites in the lin-14 3’UTR, LIN-14 protein levels are not further increased when F25B4.6/HMG-CoA synthase is inhibited. The miRNA abundance is unaffected in F25B4.6/HMG-CoA synthase RNAi. These results show that F25B4.6/HMG-CoA synthase is required for the miRISC activity, downstream of miRNA biogenesis/accumulation.

Isoprenoids, the end products of the mevalonate pathway, feed into a wide range of downstream biosynthetic pathways in additional to the better known sterol synthesis: dolichol serves as the lipid carrier of the oligosaccharide moiety for protein N-glycosylation; ubiquinone and heme A function in the electron transport chain; prenylated proteins; and isopentenyl adenine, present in position 37 of tRNAs that read codons starting with U. Addition of mevalonate to plates rescues the let-7-deficient-like phenotypes of F25B4.6/HMG-CoA synthase inactivation, but not inactivation of the miRNA Argonaute alg-1/2 by RNAi. Cholesterol does not rescue the RNAi depletion of F25B4.6/HMG-CoA synthase. This indicates that instead of cholesterol, other biosynthetic product(s) of the mevalonate pathway modulate miRNA activity. HMG-CoA reductase (encoded by F08F8.2) also acts in the miRNA pathway. Homozygous F08F8.2(tm4368) animals arrest at the L1 stage. High concentration of mevalonate rescues the homozygous F08F8.2(tm4368) animals. F08F8.2(tm4368) homozygous mutant animals growing with low mevalonate fail to express the col-19::GFP in the let-7(mg279) mutant background. Statins inhibit HMG-CoA reductase activity. When fluvastatin is added to the growth medium, let-7(mg279 animals fail to express col-19::GFP, similar to the RNAi depletion of F25B4.6/HMG-CoA synthase or the F08F8.2(tm4368) mutation. Thus in C. elegans, the non-sterol output of the mevalonate pathway modulates miRNA activity.

Even though the RNA modification output was the most attractive model for how the mevalonate pathway intersects with miRNA regulation of target mRNAs, mass spectroscopy on nuclease P1 digested C. elegans 18-28nt small RNAs (~30% are miRNAs) did not detect N6-(∆2-isopentenyl)adenosine, whereas we could easily detect it in nuclease P1 treated C. elegans tRNA. We therefore broadened the search to include cherry-picked RNAi clones targeting the genes that function in protein prenylation, tRNA isopentenyltransferase, ubiquinone and heme A biosynthesis, dolichol synthesis and protein N-glycosylation. We performed an RNAi screen for gene inactivations that cause a col-19::GFP expression defect in the let-7(mg279) mutant background. The hits that cause failure of col-19::GFP expression all function in the pathway for protein N-glycosylation. The C. elegans oligosaccharyltransferase (OST) complex that carries out protein N-glycosylation has five subunits: the catalytic subunit T12A2.2/STT-3; and four accessory subunits: T22D1.4/ribophorin I, ostd-1, ostb-1 and dad-1. Knocking down any of these genes by RNAi causes col-19::GFP expression defect in the let-7(mg279) mutant, but not in wild type, suggesting a strong genetic interaction with let-7. Inactivation of the OST complex causes de-repression of miRNA targets, with no effect on the mature miRNA levels. This indicates that the activity, rather than the biogenesis or accumulation of miRNAs, requires the N-glycosylation.

Statins block cholesterogenesis by inhibiting HMG-CoA reductase, the rate-limiting enzyme in the mevalonate pathway and are prescribed to hundreds of millions of patients to lower the risk of heart diseases. While the efficacy of statins to lower cholesterol is the general view of their molecular mechanism, the central role that mevalonate regulation of N-glycosylation to in turn mediate the function of miRNA repression of target mRNAs suggest a miRNA axis of statin treatment of heart disease (5). Significantly, a deletion of the cardiac miRNA mir-208 strongly suppresses the high blood pressure induction of fibrosis caused by the adult stage misexpression of fetal myosin heavy chain gene (6). The mevalonate has also emerged from another miRNA analysis: mir-122 is required for the replication of hepatitus C virus and statins act in the same manner as loss of mir-122 to inhibit hepatitis C replication (7-9). mir-122 mediates the recruitment of HCV replication and associated small RNA proteins such as Argonautes to lipid vesicle replication zones within cells (8). The finding that in miRNA repression of target mRNAs in animal cells engages a vesicle sorting pathway is consistent with a sorting to such organelles in miRNA regulation of target mRNAs (10-12).

RNAs linked to the proteome. The small RNA field has now collected millions of small RNAs, via library construction and high throughput DNA sequencing approaches. We have been in the descriptive phase of small RNA biology for almost a decade: for the endogenous siRNAs, there are thousands of genes represented by antisense small RNAs and millions of distinct siRNAs that tile along mRNAs represented, but no clear function has emerged. Protein cofactors for small RNAs, identified either biochemically in assays for production of siRNAs or miRNAs or piRNAs, or genetically via screens for defective small RNA function have also emerged: For example, Dicer and the proteins of the Argonaute class that mediate the maturation and nuclease guidance activities and more recently, via RNAi screens for protein cofactors of small RNA pathways in our lab and other labs that revealed many other possible components that may sort and guide small RNAs to their targets (1,2).

We believe that the clever small RNA cloning approaches used by the field have systematically missed key small RNAs that are modified at their 5’ and 3’ ends to dramatically change the ability of these RNAs to interact with the proteome and other molecules in the cell. For example, the now established 5’ ligation of dephospho-coenzyme A to RNAs allows such small RNAs to form disulphides covalently with cysteine residues on proteins (or to glutamate or aspartate residues via thioester bonds), to essentially bar code those proteins with small RNA moieties that can base pair with RNA, DNA, or other RNA-tagged proteins. Such modified proteins could then be sorted via these tags, or asssemble onto other nucleic acid scaffolds, such as the kinetochore, or scaffolds that interact with nucleic acids, such as the centrosome or cytoskeleton.

Dephospho coenzyme A has a ligatable 3’ OH. Acetyl coenzymeA as well as the synthetase that generates it is localized to the nucleus for acetylation of histones and other transcription factors. S adenosyl methionine also bearing a 3’ hydroxyl that could ligate to small RNAs. SAM is the methyl donor in a large array of methylation reactions, including methylation of histones, a connection that is not uninteresting with regard to small RNA pathways. The linkage of S adenosyl methionine or S adenosyl homocysteine to the 5’ end of small RNAs could also allow these RNAs to accompany histone methylases to the nucleus and perhaps ligate small RNAs as they transfer a methyl group. NAD. Notice that the 3’ OH of the ATP moiety could be ligated to RNA and has been shown to be ligated (4). The ligation of NAD to the 5’ end of small RNAs would allow them to participate in adenylation reactions of glutamate, aspartate and arginine, leaving the small RNA tagging the target protein. Similarly, poly ADP ribosylation could multiply tag proteins with small RNAs. Interestingly, the poly ADP polymerases pme-1 and pme-2 are the closest of all C. elegans proteins in our phylogenetic profile to the argonautes suggesting that poly ADP modification is correlated with small RNA pathways in animals, plants, fungi and protists.

Support for the idea of ligation of coenzyme A to the 5’ end of small RNAs has emerged from multiple experiments as well. Based on the concept that dephosphocoenzyme A could ligate to RNAs and to then allow them to interact with the cysteine proteome, we searched for incorporation of 35S into RNA preparations from C. elegans and S. pombe. We saw incorporation of 35S in a molecule of about 20 to 25 nt only in C. elegans, that co purifies with RNA in phenol extractions in the trireagent or SDS proteinase K digestion isolation, ethanol precipitation, and binding to RNA purification resins. We could see the expected charged tRNAs for Met and Cys in both C. elegans and S. pombe. However the 35S RNA, which we have named suRNA is not sensitive to various RNAses, suggesting that either it is highly modified or not an RNA. One concern was that sulfated glycosaminoglycans might be 35S-labeled in these preparations, though we did not expect such a narrow size range for such polysaccharide polymers. Treatment with a bacterially derived heparinase degrades the 20 to 25 nt 35S fraction band. However, the annotation of commercially bought heparinase II, isolated from Flavobacteria, is not definitive at all. It is possible that these enzymes are actually involved in disruption of small RNA pathways, especially suRNAs and that they are promiscuous for other sulfated polymers.

Even more strongly supporting the model that modified nucleotides are ligated to small RNAs is mass spectroscopy of E. coli RNA by the Liu group, who reported that succinyl coenzyme A is ligated to RNAs isolated from E. coli and other bacteria (4). Their mass spec analysis revealed a number of mystery peaks that might correspond to other ligations to RNAs, and proved that NAD is added in an analogous manner (5). The focus of their work is to reveal other enzymatic capabilities of RNAs, with major implications for the capacities in the RNA world.

We expect to find that protein elements of the centrosome for example, are ligated to siRNAs corresponding to the centromere elements of C. elegans and other organisms to mediate the binding of particular proteins to these DNA regions. We expect to find that proteins that form multisubunit complexes may use complementary small RNAs to form and disassemble complexes. Ligation of small RNAs to proteins allows the addition of a handle that can be a bar code for assembly of protein complexes and for interaction of proteins with other nucleic acids. In the same way that PCR primers allow us to bar code and preferentially amplify segments in the lab, a small RNA tag on a protein allows that protein to be recognized by other proteins bearing the complementary RNA tag.

Xenobiotic and pathogen surveillance genetics and functional genomics

Our initial genetic analysis of the C. elegans response to natural chemical toxins reveals a complex genetic pathway for xenobiotic detection, aversion, and detoxification. We already know that many of the regulatory factors we have identified in our pilot studies are conserved in humans and distinct from those being studied in mammalian pharmacogenomics, which focuses on variation in cytochromes and other enzymes that modify drugs, rather than on the identification steps in the regulatory cascade from drug detection to detoxification. The human homologues of the genes we identify promise to explain how humans respond appropriately and inappropriately to drugs, or activate drug detoxification pathways in the absence of a triggering drug, perhaps inducing a false endocrine state of poisoning. Variation in xenobiotic detection and response pathways will be most obviously important for drug responses such as toxic responses to therapeutic drugs, for example, nausea or hypersensitivity, and addictive behaviors to drugs of abuse. But we believe that such variation will be the cause of diseases as diverse as anorexia nervosa, migraine, depression and developmental defects induced by reduction of function mutations in core cellular components such as mitochondrial and nuclearly encoded mitochrondrial genes.

We view the detection of drugs as developmental biologists, with key regulatory points and checkpoints, which is quite distinct from the standard model in pharmacology. Our hypothesis is that it is the decrease in the efficiency or activity of core cellular components by drugs or mutations that is detected, not the drugs themselves. This hypothesis explains our observations that inactivation of the core cellular components by RNAi mimics the drug detoxification inductions of drugs. The assessment of the decrease in function of the core conserved cell components triggers expression of drug modification and transport genes, as well endocrine signals to suspend feeding and move to a new nutritional source. We aim to identify all of the genetic components of the core cellular component assessment systems, the endocrine systems of spreading the detoxification signal systemically, and the coupling of these stress signals to upregulation of xenobiotic detoxification systems. Our genetic analysis of how toxins are detected and which endocrine signals are produced to make the animals feel sick may identify new endocrine pathways that have human homologues. Drug development targeting those pathways could serve as adjuvants for chemotherapeutic treatment of cancer, to suppress nausea, perhaps allowing the use of higher therapeutic doses, to mention one indication. Human variation in response to drugs and to the wide range of chemicals in the normal diet may be more common than previously appreciated. The most obvious manifestation would be toxic responses in a small percentage of humans to common doses of drugs or common foods. It is also possible that aberrant constitutive upregulation of drug detoxification pathways in the absence of a toxic trigger may induce feeding dysfunctions and drug detoxification behaviors in humans bearing variation in the xenobiotic response pathways we will discover. For example, inappropriate up-regulation of drug detoxification pathways may underlie migraine headaches and anorexia. These are good candidates for diseases of inappropriate xenobiotic responses because of their strong gender bias: 3 to 10 fold more women than men suffer from migraines and anorexia. Response to xenobiotics would be predicted to be more robust in females, where a short duration of poisoning can cause catastrophic fetal development defects, whereas in a male, it can be just a day of feeling poor. The headaches and feeding suppression in such disorders would be normal outputs of a xenobiotic detoxification program, if the patient was actually ingesting a poison. But in the absence of a toxin, the response is inappropriate and induces protective measures that decrease health. Similarly, the developmental arrest phenotypes induced by decrements in essential gene activities we have shown to be a genetic program subject to suppression by mutations in the program of arrest. That is, we believe that some of the some of the symptoms induced by reduction of function mutations in core cell components may represent a genetic program of developmental arrest normally triggered by natural xenobiotic chemicals that target these components. Abrogation of these xenobiotic detection programs may paradoxically improve the health and well-being of patients bearing such mutations. The genes we have so far identified and propose to comprehensively identify may constitute drug targets for the treatment of these diseases not heretofore considered treatable without gene therapy: mitochondrial disorders, cytoskeletal disorders and other mutations in the core components of cells that cause developmental defects. We do not propose to bypass the need for mitochrondria or ribosomes, only to suppress the phenotypes of weak mutations in such systems. Weak alleles are actually very common in these genetic diseases, where the null phenotype is embryonic lethality. Finally, the drug or essential gene inactivations in C. elegans inhibit feeding and induce an aversive program. The endocrine state of these aversively stimulated animals may be homologous to the endocrine state of humans who feel unwell. As we seek genetic suppressors of this aversive behavior, an endocrinology of feeling ill would emerge. Human homologues of the endocrine signals we identify could be used to develop antagonist drugs that might treat the malaise of being ill.

Surveillance of essential cellular functions and xenobiotic detoxification in Caenorhabditis elegans

Naturally occurring antibiotics, or xenobiotics, are produced by a wide range of bacteria and fungi and target highly conserved proteins in eukaryotes and other microbes, such as the ribosome, mitochondria, and cytoskeleton. By targeting conserved RNAs and proteins, these antibiotics can disable a wide range of eukaryotic and prokaryotic competitors of the drug producing organisms. These xenobiotics cause developmental arrest or other toxic responses in many eukaryotes as well as other bacteria. In this chemical war game of measures and countermeasures, the target organisms respond to these xenobiotics through system-wide adaptive mechanisms and chemical detoxification.

We have done pilot genetic and functional genomic screens and propose more comprehensive RNAi and genetic screens and deep sequencing experiments to discover the regulatory cascade that mediate the surveillance of these toxins and their detoxification in animals. At the least ambitious level, understanding the genetic network of xenobiotic sensing and detoxification will provide insights into the pharmacokinetics of medicines, drug resistance, and responses to therapeutic chemicals as well as toxic chemicals. This is a field at its mezzanine level of sophistication. In mammals and insects, chemical detoxification pathways are activated to upregulate a battery of detoxification enzymes, including cytochrome P450, short-chain dehydrogenase/reductase, UDP-glucuronosyltransferase, and glutathione S-transferase genes. The best studied regulators of this response in mammals are the nuclear hormone receptors of the CAR and PXR class that are thought to use promiscous ligand binding domains to detect particular classes of xenobiotic chemicals to then activate these nuclear hormone receptors to directly regulate the enzymes listed above. The ligand binding domain of nuclear hormone receptors such as CAR are thought to detect suites of chemicals. This sort of model explains the detection of some chemical triggers but suffers from the problem of how the infinite number of possible chemical toxins are detected by the few NHR genes in mammals (there are 48 human NHRs).

Our hypothesis is that this mechanism for xenobiotic regulation is not general, and that instead it is the drug inhibition of core cellular components that is monitored and detected, not the drugs themselves. This model explains our observations outlined below that inactivation of the core cellular components by RNAi induces the same drug detoxification response induced by drugs. These data suggest that it is the assessment of the decrease in function of the core conserved cell components that is assessed when drugs are given, and serves to trigger drug detoxification gene expression. A signal from these core components, for example, a kinase cascade coupled to a transcription or chromatin factor, but also possibly activation of small RNAs, triggers expression of drug modification and transport genes, as well endocrine signals to not feed and move to a new nutritional source and systemically upregulate drug detoxification pathways in cells outside of the sentinel signaling cells.

How we came to the model that there is an endocrinology of core cellular component assessment: Our research over the past 15 years has revealed an insulin and TGF-beta endocrine system that controls whether the animals grow reproductively or arrest at the dauer diapause stage. Diapause arrest is an essential feature of many vertebrate and invertebrate life cycles, especially in regions with seasonal temperature and humidity extremes. Animals in diapause arrest survive temperature or humidity extremes or other stresses. For C. elegans, RNAi can be induced by feeding wild type or RNAi hypersensitive C. elegans strains any one of 19,000 isogenic E. coli strains that differ only in which of the C. elegans genes is expressed. Each dsRNA is expressed in the E. coli from a plasmid bearing converging T7 RNA polymerase promoters into which is cloned 1 kb of each C. elegans gene. There are 2700 distinct gene inactivations in C. elegans that cause developmental arrest or cessation of reproduction. The gene identity is annotated for each of these RNAi strains so that the molecular defect that causes developmental arrest is instantly revealed. The ribosome, the mitochrondrion, vacuolar H+-ATPases, the F0F1 proton channel ATPase and other core components of cells strongly emerged from this RNAi screen for developmental arrest.

A common theme to many of these cellular components is that many are targets of antibiotics produced by a range of fungi and microbes that nematodes encounter in the environment. We hypothesize that as a larvae or adult enters an environment with an antibiotic, there may be signaling pathways that detect, for example, antibiotic-induced ribosomal deficiency to trigger cessation of reproductive developmental trajectory, arrest at a particular developmental point, as well as xenobiotic protective pathways in all of the cells of the animal via an endocrine relay system. The induced stress adaptation and survival pathways would ensure that the animal could escape the antibiotic and resume reproductive development in a less toxic environment or feeding on less toxic microbes. Inhibition of translation by RNAi of translation factors may mimic the ribosomal function deficiency induced by antibiotics in the normal C. elegans ecosystem, and trigger the physiological response of developmental arrest and cessation of reproduction. Other gene inactivations also causing arrest are a vacuolar ATPase and the mitochondrial ATP synthase, also targets of natural antibiotics.

C. elegans aversion to drugs and essential gene inactivations, a nausea response.
C.elegans tend not to leave an E coli lawn or other desirable bacteria from their normal habitat which they feed on unless the animals are stressed by some aversive stimulus. As shown in the figure to the left, we noticed when working with the gene inactivations that cause developmental arrest that the animals tended to leave the lawn of bacteria targeting the core components of cells that are essential genes, unlike control E. coli strains expressing dsRNAs corresponding to other C. elegans genes that do not induce arrest. This suggested that the developmental arrest is coupled to a behavioral repertoire that includes “feeling sick” and a calculation that the source of that stress is the ingested food. Because bacteria and fungi produce a large array of xenobiotic secondary metabolites, generally products of polyketide synthase genes or non-ribosomal peptide synthesis genes which constitute 5-10% of many microbial genomes, the conclusion that any drug was likely to come from a bacteria or fungus on which the animal was feeding is reasonable. The physiological calculus of the animal may be that when, for example, the ribosome is not functioning at 100%, the most likely cause in the soil would be feeding on a bacterium or fungus that synthesizes an antiribosomal drug. Therefore cessation of feeding at that instant, and induction of dispersal behavior seeking a different source of nutrition is of high selective value.

To test if the dispersal behaviors induced by essential gene inactivation are congruent with the behaviors induced by drugs, we challenged C.elegans on a benign E. coli food source with antibiotics that inhibit distinct core components and assayed dispersal behavior. We tested drugs that target translation (Geneticin), the electron transport chain (antimycin), proteasome (Bortezomib), vacuolar ATPase (concanamycin A), ER stress (tunicamycin), oxidative stress (paraquat) and DNA damage (zeocin). Each drug induced animal dispersal from an otherwise benign bacterial lawn, reiterating the response elicited by RNAi against the corresponding protein targets. As shown in the figure to the left, these drugs stimulated dispersal within the first 4-6 hours of exposure, and as early as 1 hour.

We observed dispersal behavior in roughly one-third of the 2700 essential gene inactivations tested. Only 1%, or 30x lower frequency, of a random selection on non essential RNAi clones induced dispersal. In many cases arrested animals move significantly faster than animals on control RNAi. Animal speed and tortuosity (a metric for the degree of turning within an object’s trajectory) were quantitated using object-tracking software. Animals grown on repellant RNAi clones travelled much faster and exhibited straighter trajectories than those grown on control RNAi. These trajectories are consistent with dispersal behavior and are reminiscent of the bacterial chemotactic changes in tumbling vs straight trajectories when attracted or repelled by nutrients. We tested for the induction of a stress reporter genes by the drugs and by RNAi of genes that cause developmental arrest because these GFP fusion inductions could be used to advantage in genetic screens for poison blind mutants. Many of essential gene inactivations that induce dispersal also induce the GFP reporters responsive to ER stress (hsp-4::GFP), mitochondrial stress (hsp-6::GFP), oxidative stress (sod-3::GFP), xenobiotic and oxidative stress (gst-4::GFP) or osmotic stress (gpdh-1::GFP). There was ~20-fold enrichment for induction of stress reporters in the test gene set, with 40% of the genes tested exhibiting strong activation of at least one of the reporters, while ~1.9% of a random clone set strongly activated a reporter. These data suggest that behavioral aversion to drugs is coregulated with drug detoxification pathways indicated by the fusion genes.

The drugs also induce these fusion genes. Tunicamycin is a natural product the bacterium Streptomyces Iysosuperficus that C. elegans might naturally encounter in the soil. Tunicamycin affects the ER by inhibiting an enzyme required for N-linked glycosylation and strongly induces HSP-4, a component of the ER unfolded protein response. Antimycin is a natural product Streptomyces bacteria in the soil and inhibits the mitochondrial electon transport complex 3 by binding the cytochrome b subunit. This blocks the transfer of electrons between subunit b and subunit c, preventing ATP generation. Antimycin induces hsp-6, a component of MT UPR. One trivial explanation for the dispersal behavior of animals that are arrested is that any arrest has an associated non-feeding response. Two approaches have been used to assess this. First, if the arrest phenotype can be suppressed, it strongly favors a program of arrest and dispersal rather just a default arrest state. We have found that mutations in daf-18, the PTEN homologue of C. elegans, strongly suppress the disperal behavior of many of the arrested animals, without actually suppressing the arrest itself, pointing to an insulin pathway for the disperal behavior but not for the arrest. Secondly, our genetic analysis below identifies gene inactivations that suppress the arrest induced by drugs, again favoring a genetic program for arrest. These have not yet been tested for suppression of the dispersal behavior.

Inactivation of essential cellular activities stimulates pathogen and detoxification responses

Because bacterial pathogens are known to stimulate food-avoidance behavior in C. elegans, the observation that inactivation of cellular processes using toxins or RNAi produced a similar behavioral response suggests an adaptable mechanism for toxin and pathogen detection mediated by cellular surveillance pathways in the host. We reasoned that if animals use internal physiologic cues to recognize the presence of pathogens, then perhaps RNAi of essential cellular components would trigger physiologic defenses associated with pathogen infection or toxin exposure – in the absence of pathogens or toxins.

We examined the expression of several GFP reporters previously shown to represent activation of innate immune programs: clec-60 (a C-type lectin/CUB domain protein induced by S. aureus and M. nematophilum), F35E12.5 (a CUB domain protein induced by Y. pestis, M.nematophilum and P. aeruginosa) , and nlp-29 (a conserved glycine/tyrosine-rich antimicrobial peptide induced by D. coniospora, S. marcescens and wounding). Finally, irg-1::GFP is a reporter induced by P. aeruginosa and cadmium poisoning whose expression in response to pathogen exposure requires ZIP-2, a bZIP transcription factor. Induction of each pathogen reporter was analyzed against a panel of ~100 gene inactivations representing the major functional classes identified in our aversion screen and a random panel of genes from the whole genome RNAi library. For all 4 pathogen-response genes, we observed a significantly elevated frequency of activation by the aversion gene set relative to the control set. In general, the three intestinal reporters were induced by RNAi against most functional classes, with most potent induction following disruption of protein synthesis, metabolic and mitochondrial functions.

If host surveillance pathways detect the presence of chemical toxins by monitoring core cellular activities, then RNAi-mediated disruption of these activities might also stimulate drug detoxification responses, such as cytochrome P450 and glutathione S-transferase genes. We assayed GFP transcriptional reporters for induction of xenobiotic detoxification enzymes, cyp-35B1 (an intestinally-expressed cytochrome P450 oxidase) and gst-4 (a glutathione S-transferase induced by drugs and toxins). Each of these reporters was induced at a frequency ~4-fold above background (~40% of aversion-inducing essential gene inactivations tested induced cyp-35B1::GFP and ~20% induced gst-4::GFP, p<0.0001, chi-squared test).

An endocrine system for assessment of core cellular components
Our analysis suggests that the integrity of these core cellular components may be assessed in cells that tend to be the most exposed to the environment, and that a signaling pathway to endocrine control of development and reproduction may operate. For example, we have found that there are sentinel signaling cells: the intestine where microbes are first encountered in an intimate way and sensory neurons that might “probe” the antibiotic environment before the rest of the cells of the animal are inhibited. These sensory cells are likely to be endocrine beacons to the rest of the animal. We used a collection of strains in which gene knockdowns can be accomplished within a single, restricted tissue using the same technique of feeding animals bacteria expressing dsRNA against target genes. The strain backgrounds used for these experiments are fully defective for RNAi because they lack the necessary gene (either rde-1 or sid-1). Only the tissue(s) in which gene function is restored (using tissue-specific promoters to rescue rde-1 or sid-1 expression) are competent for gene knockdown when fed RNAi against a given target. In addition, we a mutant strain proficient for RNAi in the germline but not in somatic tissue (rrf-1) due to differing mechanisms of RNAi in these tissue types. Inactivation of core cell components in each tissue tested – the hypodermis, the intestine, the gonad and body wall muscle – is competent to trigger dispersal. This suggests the existence of an endocrine system for response to essential gene inactivation.

The molecular mechanism of xenobiotic detection to induce xenobiotic protection programs and developmental arrest: We interpret developmental arrest differently than most who work on essential genes. Usually such arrests or sterility are considered the consequence of a loss of an essential cellular component; that the animal cannot develop past a developmental milestone in the absence of the particular gene product. However, another view of such developmental arrests is that they are a “programmed” response to a deficiency in a key function, and that active signaling pathways mediate the arrest point, as a sort of “developmental checkpoint”. This view is motivated by the finding that arrest at the dauer stage is an active process and can be abrogated by mutations in dauer defective genes such as the FoxO factor DAF-16 or the Smad factor DAF-3 or the nuclear hormone receptor DAF-12. Importantly such an arrest program interpretation depends on two key attributes: 1. The arrest should be reversible. We have observed that when drug is removed in many cases, the arrest is reversible. 2. There should be mutations that disable the arrest program. This is also established below.

We have used the failure to induce the ER or mitochondrial or other stress GFP fusion genes under drug or stressed conditions to derive strains of C. elegans that are “blind” to mitochrondrial dysfunction or ribosomal dysfunction, and therefore do not induce these GFP fusion genes (outlined in the figure to the left). We can then ask if a failure to induce these genes makes the strains more or less sensitive to these drugs, that is disrupts the normally associated induction of drug resistance, allowing us to study the genetic pathways of xenobiotic surveillance and detoxification. In this way, we will discern the signaling pathway, both within cells and between cells for xenobiotic responses. We may find that animals deficient in these stress sensory or response execution pathways have major defects in the detoxification of xenobiotics or in the responses that allow survival when intoxicated.

We first did pilot screens starting with cherry picked libraries of sublibraries, and propose to ramp up to full genome screens in this proposal. Our first candidate genes for signaling from essential gene inactivation to xenobiotic resistance emerged from a survey of gene inactivations from four 100 to 300 gene candidate libraries. So far from these pilot screens, shown in the table to the left, we have identified 20 gene inactivations that fail to activate the drug induced reporter genes. These genes come from all three candidate libraries. The genes identified by these inactivations are candidates for the genetic pathways that transduce deficiencies in ribosomal or mitochrondrial function to endocrine pathways for developmental arrest.

The key difference between how we have come to view xenobiotic detection and how the mammalian or insect drug detoxification/insecticide resistance field views the problem: Our focus on drug resistance is distinct from the usual interest in resistance or sensitivity one drug or pathway. We are most interested in mutations or gene inactivations that broadly disable response to inactivation of the ribosome or of the mitochondion but not in mutations that make the target protein of the drug compromised for binding, for example by mutating a key amino acid residue for drug binding. Find the drug receptor is the usual goal of drug resistance genetics, though the insecticide resistance field has broadened the goals to discover gene amplifications that mediate resistance (2). In contrast, we wish to identify the signaling components that sense the ribosome or mitochrondrial or cytoskeletal integrity and signal from these assessments to generalized drug detoxification pathways. So our expectation is that many of the genes we identify will make animals hypersensitive or resistant to more than one class of drug.

A signal from these core components, for example, most likely a kinase cascade to a transcription or chromatin factor, but also possibly activation of small RNAs, triggers expression of drug modification and transport genes, as well endocrine signals to not feed and move to a new nutritional source. This is different than the problem posed by most of the field of toxicology: how do the transcriptional regulatory factors such as the nuclear hormone receptor CAR recognize the infinite number of possible chemicals in the toxin world. If toxins are recognized by their inhibition of the ribosome or mitochondrion or other core cell components, the only recognition needed is that they are drugs that cause a decrement in core component function. That is only the function of the core elements must have a checkpoint control.