RNA interference (RNAi) plays crucial roles in regulating many important biological processes using short interfering RNAs (siRNAs). In C. elegans, siRNAs, 22G-RNAs, are generated using template mRNAs and non-coding RNAs by a complex containing RNA-dependent RNA Polymerase (RdRP) and Dicer Related Helicase 3 (DRH-3). Our previous observations indicated that DRH-3 is essential to the biogenesis of siRNAs mapped to the 5' UTRs and the protein coding regions but not to the very 3' end of 3' UTRs. Therefore, in the
drh-3 mutants, 22Gs are still being made at the 3' UTRs of RNAs. We speculate that siRNA generation starts from the 3' UTRs of target RNAs using a machinery containing DRH-3 and RdRPs, and DRH-3 is required for the translocation of this machinery towards the 5' end of target RNAs to make more siRNAs. We propose two models to explain the function of DRH-3. In model 1, DHR-3 unwinds the siRNAs generated by RdRP from template RNAs, and facilitates the loading of siRNAs into Argonautes including WAGO-1 and/or WAGO-9. Without DRH-3, siRNAs are only generated at the 3' UTRs but cannot be unwound and loaded into Argonautes. As a result, RdRP cannot move along template RNAs and the cloned 22Gs are localized to the 3' UTR of RNAs. In model 2, DRH-3 is required to unwind mRNA secondary structure ahead of RdRPs to allow the translocation of RdRPs. Without DRH-3, the siRNAs are still only generated in 3' UTRs but can be loaded into Argonautes. To distinguish these two models, we generated two
drh-3 mutants with a transgene that contains either a FLAG-tagged WAGO-1 or FLAG-tagged WAGO-9, both of which binds DRH-3 dependent siRNAs in wild type cells. Using immunoprecipitation followed by high-throughput sequencing, we aim to detect whether the 3'UTR-siRNAs in the
drh-3 mutants bind these two Argonautes, as predicted by model 1. We have generated these strains, performed the immunoprecipitation, and created the high-throughput sequencing library. We are performing high-throughput sequencing and bioinformatics analysis.