The Nuclear Argonaute NRDE-3 Contributes to Transitive RNAi inCaenorhabditis elegans

Abstract

The Caenorhabditis elegans nuclear RNA interference defective (Nrde) mutants were identified by their inability to silence polycistronic transcripts in enhanced RNAi (Eri) mutant backgrounds. Here, we report additional nrde-3-dependent RNAi phenomena that extend the mechanisms, roles, and functions of nuclear RNAi. We show that nrde-3 mutants are broadly RNAi deficient and that overexpressing NRDE-3 enhances RNAi. Consistent with NRDE-3 being a dose-dependent limiting resource for effective RNAi, we find that NRDE-3 is required for eri-dependent enhanced RNAi phenotypes, although only for a subset of target genes. We then identify pgl-1 as an additional limiting RNAi resource important for eri-dependent silencing of a nonoverlapping subset of target genes, so that an nrde-3; pgl-1; eri-1 triple mutant fails to show enhanced RNAi for any tested gene. These results suggest that nrde-3 and pgl-1 define separate and independent limiting RNAi resource pathways. Limiting RNAi resources are proposed to primarily act via endogenous RNA silencing pathways. Consistent with this, we find that nrde-3 mutants misexpress genes regulated by endogenous siRNAs and incompletely silence repetitive transgene arrays. Finally, we find that nrde-3 contributes to transitive RNAi, whereby amplified silencing triggers act in trans to silence sequence-similar genes. Because nrde-dependent silencing is thought to act in cis to limit the production of primary transcripts, this result reveals an unexpected role for nuclear processes in RNAi silencing. GENETIC analysis of RNA interference (RNAi) in Caenorhabditis elegans initially identified and characterized genes and activities important for post-transcriptional gene silencing (PTGS) in response to exogenous double-stranded RNA (dsRNA) (Tabara et al. 1999(Tabara et al. , 2002. Because these gene products target mature RNAs (mRNAs), they were presumed to act in the cytoplasm . However, a genetic screen for mutants specifically defective for pan-operon RNAi silencing (Bosher et al. 1999) identified genes important for transcriptional gene silencing (TGS) processes that operate in the nucleus (Guang et al. 2008(Guang et al. , 2010Burkhart et al. 2011;Burton et al. 2011). These genes were termed nuclear RNAi defective (nrde).The best-characterized nrde is NRDE-3, an Argonaute that shuttles secondary short-interfering RNAs (siRNAs) from the cytoplasm to the nucleus (Guang et al. 2008). When siRNA are absent or reduced, NRDE-3 is primarily detected in the cytoplasm, and when siRNAs are abundant, NRDE-3 is readily detected in the nucleus. In the nucleus, the siRNAbound NRDE-3 forms a complex with the nuclear restricted NRDE-1, -2, and -4 (Burton et al. 2011). The associated siRNA guides the NRDE complex to cognate nascent mRNA transcripts, which impedes RNA polymerase II transcription elongation and further initiates histone methylation-dependent TGS (Guang et al. 2010;Burton et al. 2011). This RNA-directed epigenetic mechanism also enables multigenerational silencing ; the recently ident...

Movement of regulatory RNA between animal cells

“…RDE-1 – see Grishok, 2013 for comprehensive review), we can conclude that this gene is also required for silencing by mobile RNAs (if any) derived from ingested or injected dsRNA. Different genes can be required for ingested dsRNA to silence different targets (Zhuang et al, 2013). For example, the nuclear Argonaute NRDE-3 but not the P-granule component PGL-1 is required to silence lir-1 , and PGL-1 but not NRDE-3 is required to silence unc-73 .…”

Nuclear RNAi Contributes to the Silencing of Off-Target Genes and Repetitive Sequences in Caenorhabditis elegans

“…This effect could be partially explained by a competition model in which the exogenous RNAi pathway competes with the endogenous RNAi pathway for limiting factors (Lee et al 2006;Yigit et al 2006). Indeed, the overexpression of SAGO-1, SAGO-2, or NRDE-3 enhances feeding RNAi, indicating that some secondary Argonaute proteins could be the limiting factors (Yigit et al 2006;Zhuang et al 2013). On the other hand, our data suggest that at least some of the enhanced RNAi effect could arise from off-target silencing, particularly for dsRNAs targeting large gene families.…”

Early Developmental Exposure to dsRNA Is Critical for Initiating Efficient Nuclear RNAi in C. elegans

“…Interestingly, nuclear RNAi is considerably more potent in the F1 progeny than in the P0 worms that were initially exposed to dsRNA (Burton et al, 2011, Zhuang et al, 2013). For example, the phenotype of dpy-11 RNAi is much stronger in the F1 progeny than in the P0 generation, and deposition of H3K9 methylation at the dpy-11 locus is also much higher in the F1 generation than the P0 generation (Burton et al, 2011).…”