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.