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These authors contributed equally % Corresponding author: Eric A Miska: eam29@cam.ac.uk, +44-1223-334088, https://orcid.org/0000-0002-4450-576X 1 Manuscript ABSTRACT PIWI-interacting RNAs (piRNAs) are genome-encoded small RNAs that regulate germ cell development and maintain germline integrity in many animals. Mature piRNAs engage Piwi Argonaute proteins to silence complementary transcripts, including transposable elements and endogenous genes. piRNA biogenesis mechanisms are diverse and remain poorly understood. Here, we identify the RNA Polymerase II (RNA Pol II) core subunit RPB-9 as required for piRNA-mediated silencing in the nematodeCaenorhabditis elegans . rpb-9 mutants fail to initiate heritable piRNA-mediated gene silencing. Furthermore, we show that RPB-9 is required to repress two DNA transposon families and a subset of somatic genes in the C. elegans germline. We provide genetic and biochemical evidence that RPB-9 is required for piRNA biogenesis. We demonstrate that RPB-9 acts to promote transcriptional elongation/termination at endogenous piRNA loci. We conclude that as a part of its rapid evolution the piRNA pathway has co-opted another ancient machinery, this time for high-fidelity transcription.al., 2019) at the Ruby motif. Transcribed piRNA precursors are 26 to 29 nucleotides (nt) in length and carry a 5′ 7-methylguanylate cap (Goh et al., 2014;Gu et al., 2012;Weick et al., 2014) . After transcription, precursors are promptly exported out of the nucleus, possibly by PID-1 (de Albuquerque et al., 2014) , which is predicted to have both nuclear localization and export signals, and later bound by the PETISCO/PICS complexes (Cordeiro Rodrigues et al., 2019;Zeng et al., 2019) , which might be involved in 5′ end precursor processing, including decapping and 5′ end trimming. Subsequent 3′-end trimming is mediated by the exonuclease PARN-1 (Tang et al., 2016) . The resulting mature C. elegans piRNAs are 21 nt small RNAs with a 5′ uracil bias (hence called 21U-RNAs). They also possess a 5′ monophosphate and a 3′ hydroxyl group at their extremities, and are post-transcriptionally 2′-O methylated at the 3′ end by the HENN-1 enzyme (Kamminga et al., 2010;Montgomery et al., 2012) .Mature piRNAs bind to PIWI subfamily proteins of the AGO family and guide them to complementary target transcripts. Two PIWI proteins with germline-restricted expression, PRG-1 and PRG-2, have been identified in C. elegans , although only PRG-1 is required for maintaining wild-type piRNA populations (Batista et al., 2008;Das et al., 2008;Wang and Reinke, 2008) . These proteins localize to P-granules, specialized germline-specific phase-separated perinuclear structures that are the main site of piRNA-mediated post-transcriptional gene silencing activities (Updike and Strome, 2010) .Unlike in other animals, where PIWI/piRNA complexes can silence their targets via a PIWI endonuclease "slicing" activity, PRG-1/piRNA complexes in C. elegans silence their targets by triggering their degradation indirectly. The imperfect base pairing ...
These authors contributed equally % Corresponding author: Eric A Miska: eam29@cam.ac.uk, +44-1223-334088, https://orcid.org/0000-0002-4450-576X 1 Manuscript ABSTRACT PIWI-interacting RNAs (piRNAs) are genome-encoded small RNAs that regulate germ cell development and maintain germline integrity in many animals. Mature piRNAs engage Piwi Argonaute proteins to silence complementary transcripts, including transposable elements and endogenous genes. piRNA biogenesis mechanisms are diverse and remain poorly understood. Here, we identify the RNA Polymerase II (RNA Pol II) core subunit RPB-9 as required for piRNA-mediated silencing in the nematodeCaenorhabditis elegans . rpb-9 mutants fail to initiate heritable piRNA-mediated gene silencing. Furthermore, we show that RPB-9 is required to repress two DNA transposon families and a subset of somatic genes in the C. elegans germline. We provide genetic and biochemical evidence that RPB-9 is required for piRNA biogenesis. We demonstrate that RPB-9 acts to promote transcriptional elongation/termination at endogenous piRNA loci. We conclude that as a part of its rapid evolution the piRNA pathway has co-opted another ancient machinery, this time for high-fidelity transcription.al., 2019) at the Ruby motif. Transcribed piRNA precursors are 26 to 29 nucleotides (nt) in length and carry a 5′ 7-methylguanylate cap (Goh et al., 2014;Gu et al., 2012;Weick et al., 2014) . After transcription, precursors are promptly exported out of the nucleus, possibly by PID-1 (de Albuquerque et al., 2014) , which is predicted to have both nuclear localization and export signals, and later bound by the PETISCO/PICS complexes (Cordeiro Rodrigues et al., 2019;Zeng et al., 2019) , which might be involved in 5′ end precursor processing, including decapping and 5′ end trimming. Subsequent 3′-end trimming is mediated by the exonuclease PARN-1 (Tang et al., 2016) . The resulting mature C. elegans piRNAs are 21 nt small RNAs with a 5′ uracil bias (hence called 21U-RNAs). They also possess a 5′ monophosphate and a 3′ hydroxyl group at their extremities, and are post-transcriptionally 2′-O methylated at the 3′ end by the HENN-1 enzyme (Kamminga et al., 2010;Montgomery et al., 2012) .Mature piRNAs bind to PIWI subfamily proteins of the AGO family and guide them to complementary target transcripts. Two PIWI proteins with germline-restricted expression, PRG-1 and PRG-2, have been identified in C. elegans , although only PRG-1 is required for maintaining wild-type piRNA populations (Batista et al., 2008;Das et al., 2008;Wang and Reinke, 2008) . These proteins localize to P-granules, specialized germline-specific phase-separated perinuclear structures that are the main site of piRNA-mediated post-transcriptional gene silencing activities (Updike and Strome, 2010) .Unlike in other animals, where PIWI/piRNA complexes can silence their targets via a PIWI endonuclease "slicing" activity, PRG-1/piRNA complexes in C. elegans silence their targets by triggering their degradation indirectly. The imperfect base pairing ...
Epigenetic inheritance contributes fundamentally to transgenerational physiology and fitness. Mechanistic understanding of RNA-mediated chromatin modification and transgenerational epigenetic inheritance, which in C. elegans can be triggered by exogenous double-stranded RNA (exo-dsRNA) or facilitated by endogenous small interfering RNAs (endo-siRNAs), has mainly been limited to the post-initiation phases of silencing. Indeed, the dynamic process by which nuclear RNAi engages a transcriptionally active target, before the repressive state is stably established, remains largely a mystery. Here we found that the onset of exo-dsRNA-induced nuclear RNAi is a transgenerational process, and that establishment requires SET-32, one of the three putative histone methyltransferases (HMTs) that are required for H3K9me3 deposition at the nuclear RNAi targets. We also performed multigenerational whole-genome analyses to examine the establishment of silencing at endogenous targets of germline nuclear RNAi. The nuclear Argonaute protein HRDE-1 is essential for the maintenance of nuclear RNAi. Repairing a loss-of-function mutation in hrde-1 by CRISPR restored the silencing of endogenous targets in animals carrying wild type set-32. However, for numerous endogenous targets, repairing the hrde-1 mutation in a set-32;hrde-1 double mutant failed to restore their silencing states in up to 20 generations after the hrde-1 repair, using a similar genome editing approach. We found that despite a prominent role in the establishment of silencing, however, set-32 is completely dispensable for the maintenance of silencing once HRDE-1-dependent gene repression is established. Our study indicates that: 1) establishment and maintenance of siRNA-guided transcriptional repression are two distinct processes with different genetic requirements; and 2) the rate-limiting step of the establishment phase is a transgenerational, chromatin-based process. In addition, our study reveals a novel paradigm in which a heterochromatin factor primarily functions to promote the establishment of transgenerational silencing, expanding mechanistic understanding of the well-recognized role of heterochromatin in epigenetic maintenance.
23In several species, Piwi/piRNA genome silencing defects lead to immediate sterility 24 accompanied by heterochromatin dysfunction and transposon-induced genomic 25 instability, which may cause Piwi mutant sterility. In C. elegans, Piwi pathway mutants 26 transmit a heritable stress through germ cells that induces sterility after growth for 27 several generations. We found that sterile Piwi pathway mutant germ cells displayed 28 inconsistent increases in DNA damage but consistently altered perinuclear germ 29 granules that are known to promote fertility. Germ granule dysfunction did not elicit 30 transposon expression but was sufficient to induce multiple phenotypes found in sterile 31 Piwi silencing mutants, including germline atrophy and regrowth. Furthermore, loss of 32 the germ granule component PGL-1 accelerated sterility in response to deficiency for 33 prg-1/Piwi. Restoration of germ granule function to sterile pgl-1 mutants restored their 34 fertility. Together, our results suggest that germ granule defects may promote an adult 35 reproductive arrest phenotype that is responsible for Piwi/piRNA mutant sterility.36 37
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