The PIWI-interacting RNA (piRNA) pathway in Caenorhabditis elegans engages thousands of endogenously encoded piRNAs within perinuclear nuage (P-granules) to scan RNAs exiting the nucleus. Sufficient base-pairing between a piRNA and a target RNA elicits a silencing response that is maintained transgenerationally by worm-specific Argonaute (WAGO) pathways following recruitment of RNA-dependent RNA polymerases (RdRPs) which synthesize 22 nt RNA species with a bias for a 5' guanosine (22G-RNAs). We previously described ZNFX-1 as a factor involved in piRNA-triggered RNA-induced epigenetic silencing (RNAe); ZNFX-1 was also found to engage the CSR-1 pathway which targets endogenous genes to protect them from silencing. Our study identified ZNFX-1 as being involved in positioning of RdRPs along small RNA-targeted transcripts to promote uniform 22G-RNA production to prevent bias for 5' targeting of mRNAs. Here we report that ZNFX-1 acts in parallel with PRG-1 to affect epigenetic inheritance of signals for both CSR-1 and WAGO target genes; small RNA sequencing shows us that 22G-RNAs targeting WAGO-target genes are strongly reduced in
prg-1;
znfx-1 double mutants relative to either single mutant or wild-type, and the double mutant sees increased expression of transposases for transposons Tc1 and Tc3 as shown by quantitative PCR (qPCR). Some small RNA targets whose targeting 22G-RNAs are depleted in a
prg-1 single mutant see increased small RNA production in
prg-1;
znfx-1, which may be the result of these genes becoming targets for CSR-1 rather than WAGOs. Furthermore, we see that unlike
znfx-1 single mutants which are defective in RNAi inheritance,
prg-1;
znfx-1 double mutants can inherit RNAi, consistent with the idea that loss of endogenous small RNAs frees up small RNA machinery to respond to an exogenous RNAi trigger, also suggesting that PRG-1 activity negatively regulates, and ZNFX-1 function is dispensable for, RNAi inheritance. Moving forward, we will immunoprecipitate Argonautes in the CSR and WAGO pathways and sequence the small RNA populations being loaded into these Argonautes to better understand how loss of PRG-1 and ZNFX-1 activity affects endogenous small RNA pathways, and we will sequence small RNAs in animals grown on RNAi to develop a clearer picture of how these factors act in RNAi inheritance. We will also use DNA and RNA fluorescence in-situ hybridization (FISH) to look at spatial regulation of RNAs undergoing desilencing in these mutants.