Academic journal article Genetics

The Nuclear Argonaute NRDE-3 Contributes to Transitive RNAi in Caenorhabditis Elegans

Academic journal article Genetics

The Nuclear Argonaute NRDE-3 Contributes to Transitive RNAi in Caenorhabditis Elegans

Article excerpt

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, 2002). Because these gene products target mature RNAs (mRNAs), they were presumed to act in the cytoplasm (Fire et al. 1998). 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, 2010; Burkhart 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 (Gu et al. 2012); the recently identified germline nuclear Argonaute HRDE-1 functions like the soma-restricted NRDE-3 to enable this multigenerational silencing (Buckley et al. 2012). hrde-1 is also important for germline immortality. As these enlightening studies on mechanisms and downstream endogenous roles of nuclear RNAi have progressed, broader identification of other nuclear RNAi-dependent upstream processes and their interactions with cytoplasmic RNAi therefore becomes ever more important.

Enhanced RNAi (Eri) mutant backgrounds have been important for the discovery and characterization of nrde functions. Eri mutants identify genes important for the production or stability of endogenous short-interfering RNAs (endo-siRNAs) (Simmer et al. 2002; Kennedy et al. 2004; Duchaine et al. 2006; Fischer et al. 2008; Pavelec et al. 2009). Genes complementary to endo-siRNAs are misregulated in Eri mutants (Gent et al. 2010). Interestingly, these endo-siRNAs also make up the bulk of siRNAs associated with NRDE-3 in vivo (Guang et al. …

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