In 1993 Victor Ambros and colleagues discovered the first small regulatory RNA, lin-4. Since that time andthe subsequent discovery of RNA interference (RNAi) and small interfering RNAs (siRNAs) by Andy Fire, CraigMello, and David Baulcombe, the small regulatory RNA field has expanded in directions no one could havepredicted. We now know that most eukaryotic cells express a wide variety of endogenous small regulatory RNAsthat function in a remarkably wide range of biological processes, including but not limited to: heterochromatinformation, developmental timing, defense against parasitic nucleic acids, and genome rearrangement. Themechanistic underpinnings of RNAi are broadly conserved across eukaryotes. dsRNAs are clesaved by Dicer-likeenzymes into short 20-25 nucleotide RNAs 1. These small regulatory RNAs associate with a conserved family ofproteins termed the Argonautes (Agos) 2. Together, Ago proteins, and their associated small RNAs, negativelyregulate gene expression by recognizing and inhibiting complementary nucleic acids. Our long-term goal is to understand the how and why of small RNAs and RNAi. Towards this goal we haveconducted the first forward genetic screen in metazoans seeking to identify factors required for small RNA-mediated silencing in the nucleus (nuclear RNAi). To date our screen has identified two evolutionarily conservedfactors, nuclear RNAi defective-2 (nrde-2) and nrde-3, which are required for nuclear RNAi. nrde-3 encodes anAgo protein that transports siRNAs from the cytoplasm to the nucleus 3. nrde-2 encodes an evolutionarilyconserved protein that associates with NRDE-3 in the nucleus and is directed by NRDE-3/siRNAribonucleoprotein complexes to nascent transcripts that have been targeted by RNAi. Our data indicate that smallRNAs, acting in conjunction with NRDE-2 and NRDE-3, direct a novel mode of gene regulation: termination ofRNA Polymerase II transcription. These data suggest that our genetic screen is targeting a dedicated nuclearsilencing pathway and hint that the nuclear silencing pathway we are defining represents a novel and conservedmode of gene regulation. The experiments we propose in this grant are designed to identify and characterizeadditional components of the nuclear RNAi pathway, unravel the mechanism of nuclear RNAi, and begin toelucidate the raison d' tre of small RNA-mediated gene silencing in metazoan nuclei. These experiments willprovide a framework for us, and others, to ask if these processes are mechanistically conserved in mammals. Initial successes utilizing siRNAs to target oncogenic and viral mRNAs have generated excitement thatsiRNAs may be utilized eventually to treat human disease 4. siRNA treatment could theoretically be used to down-regulate any RNA molecule, either foreign or endogenous, present within the human body. Prior to the rationaluse of siRNAs in the treatment of human disease, however, it is essential that we understand: how small RNAsare generated, how and where small RNAs function, the specificity of small RNA-driven silencing, and the role ofsmall RNAs in endogenous biological processes. Our research is addressing these questions.
Project RelevancesiRNA therapeutics may eventually be used to regulate gene expression in humans. Prior to the rational use ofsiRNAs in the treatment of human disease; however; it is essential that we understand: how small RNAs aregenerated; how and where small RNAs function; the specificity of small RNA-driven silencing; and the role ofsmall RNAs in endogenous biological processes. Our research is addressing these questions.
| Perales, Roberto; Pagano, Daniel; Wan, Gang et al. (2018) Transgenerational Epigenetic Inheritance Is Negatively Regulated by the HERI-1 Chromodomain Protein. Genetics 210:1287-1299 |
| Wan, Gang; Fields, Brandon D; Spracklin, George et al. (2018) Spatiotemporal regulation of liquid-like condensates in epigenetic inheritance. Nature 557:679-683 |
| Spracklin, George; Fields, Brandon; Wan, Gang et al. (2017) The RNAi Inheritance Machinery of Caenorhabditis elegans. Genetics 206:1403-1416 |
| Juang, Bi-Tzen; Gu, Chen; Starnes, Linda et al. (2013) Endogenous nuclear RNAi mediates behavioral adaptation to odor. Cell 154:1010-1022 |
| Gu, Sam Guoping; Pak, Julia; Guang, Shouhong et al. (2012) Amplification of siRNA in Caenorhabditis elegans generates a transgenerational sequence-targeted histone H3 lysine 9 methylation footprint. Nat Genet 44:157-64 |
| Buckley, Bethany A; Burkhart, Kirk B; Gu, Sam Guoping et al. (2012) A nuclear Argonaute promotes multigenerational epigenetic inheritance and germline immortality. Nature 489:447-51 |
| Burton, Nick O; Burkhart, Kirk B; Kennedy, Scott (2011) Nuclear RNAi maintains heritable gene silencing in Caenorhabditis elegans. Proc Natl Acad Sci U S A 108:19683-8 |
| Burkhart, Kirk B; Guang, Shouhong; Buckley, Bethany A et al. (2011) A pre-mRNA-associating factor links endogenous siRNAs to chromatin regulation. PLoS Genet 7:e1002249 |
| Guang, Shouhong; Bochner, Aaron F; Burkhart, Kirk B et al. (2010) Small regulatory RNAs inhibit RNA polymerase II during the elongation phase of transcription. Nature 465:1097-101 |
| Guang, Shouhong; Bochner, Aaron F; Pavelec, Derek M et al. (2008) An Argonaute transports siRNAs from the cytoplasm to the nucleus. Science 321:537-41 |
