Argonaute-siRNA loading via the RNA binding protein RDE-4 in C. elegans

Small RNAs associate with Argonaute proteins to control gene expression, impacting a wide range of cellular processes, such as antiviral defense, transposon silencing, and development. Plants and animals typically have several classes of small RNAs, along with multiple Argonautes. These Argonautes often confer distinct functionality to the various classes of small RNAs. But how small RNAs are selectively loaded into the appropriate Argonaute is not well understood. Small interfering RNAs (siRNAs) and microRNAs (miRNAs) are two classes of small RNAs commonly generated from double-stranded RNA (dsRNA) precursors by the endoribonuclease Dicer, although not all siRNAs or miRNAs are produced in this manner. dsRNA-derived siRNAs are often cleaved from fully base-paired precursors, whereas miRNAs typically originate from partially base-paired hairpins. In C. elegans, Dicer/DCR-1 processing of siRNAs and a related dsRNA-derived small RNA class known as 26G-RNAs is mediated by the dsRNA-binding protein RDE-4. In the study associated with this data, we used small RNA high-throughput sequencing to show that RDE-4 also promotes loading of siRNAs but not miRNAs into the Argonaute RDE-1, but not ALG-1, and loading of 26G-RNAs into the Argonaute ERGO-1, but not into the two paralogous Argonautes ALG-3 and ALG-4. Although ALG-3/4 associated 26G-RNAs do not require RDE-4 for Argonaute loading, the levels of these small RNAs are strongly reduced in rde-4 mutants. Therefore, RDE-4 functions in both dsRNA processing and selective loading of small RNAs into specific Argonautes. Our findings reveal a role for RDE-4 as a critical determinant of small RNA loading specificity and provide insight into the mechanisms by which small RNAs are selectively paired with their corresponding Argonaute proteins. Overall design: High-throughput small RNA sequencing was performed on rde-4 and rde-1 mutant strains, non-mutant controls, and transgenic strains expressing GFP or HA fusions to Argonaute proteins. The animals were either left untreated or subjected to RNAi targeting oma-1 and/or nrfl-1 ord dcr-1. RNA was extracted from cell lysates or Argonaute co-immunoprecipitates from C. elegans cultured at 20°C. The resulting small RNA data was analyzed using the tinyRNA pipeline.