Knittel, T. L.; Montgomery, B. E.; Sprister, R. A.; Magelky, C. N.; Smith, M. J.; Soto-Ojeda, M.; Guthrie, M.; Phillips, C. M.; Montgomery, T.

Small RNAs, such as microRNAs (miRNAs) and small interfering RNAs (siRNAs), 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. miRNAs and siRNAs are typically generated from double-stranded RNA (dsRNA) precursors by the endoribonuclease Dicer. siRNAs are often processed from fully base-paired precursors derived from various endogenous and exogenous sources, whereas miRNAs typically originate from genetically encoded partially base-paired hairpins. In C. elegans, Dicer/DCR-1 processing of siRNAs and a related small RNA class, known as 26G-RNAs, is mediated by the dsRNA-binding protein RDE-4. Here, we show that RDE-4 also facilitates loading of siRNAs (but not miRNAs) into the Argonaute RDE-1, but not into ALG-1, and loading of 26G-RNAs into the Argonaute ERGO-1. Although we do not find evidence that ALG-3/4 associated 26G-RNAs require RDE-4 for Argonaute loading, their 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 their corresponding Argonautes.