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

Small RNAs, such as small interfering RNAs (siRNAs) and microRNAs (miRNAs), associate with Argonaute proteins to control gene expression, impacting a wide range of cellular processes, including antiviral defense, transposon silencing, and development.1 Plants and animals have several classes of small RNAs, along with multiple Argonautes that often confer distinct functionality to these small RNAs.2,3 But how small RNAs are selectively loaded into the appropriate Argonaute is not well understood. siRNAs and miRNAs are typically generated from double-stranded RNA (dsRNA) precursors by the endoribonuclease Dicer.4 siRNAs are often processed from extensively base-paired precursors arising from various endogenous and exogenous sources, whereas miRNAs normally originate from genetically encoded, partially base-paired hairpins.1 In Caenorhabditis elegans, Dicer/DCR-1 processing of siRNAs and a related small RNA class called 26G-RNAs is mediated by the dsRNA-binding protein RDE-4.5,6,7 Here, we show that RDE-4 also facilitates the preferential loading of siRNAs into the RNA interference (RNAi) pathway Argonaute RDE-1, thereby promoting secondary siRNA amplification and facilitating an effective RNAi response. RDE-4 is also required for loading 26G-RNAs into the Argonaute ERGO-1; however, we do not find evidence that it plays a similar role in loading 26G-RNAs into ALG-3. Nonetheless, ALG-3/4 class 26G-RNA levels are strongly reduced in rde-4 mutants, indicating that RDE-4 is broadly required for their formation or stability. 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 the correct Argonautes.