A low-abundance class of Dicer-dependent siRNAs produced from a variety of features in C. elegans

Canonical small interfering RNAs (siRNAs) are processed from double-stranded RNA (dsRNA) by the endoribonuclease Dicer. siRNAs are found in plants, animals, and some fungi where they associate with Argonautes to direct RNA silencing. In Caenorhabditis elegans, some endogenous small RNAs, such as 22G- RNAs and 26G-RNAs, share certain attributes with canonical siRNAs but exhibit unique characteristics known only to occur in nematodes. For instance, 22G-RNAs do not originate from dsRNA and are not processed by Dicer, whereas 26G-RNAs require Dicer but lack the typical duplex intermediate with symmetrical 3'-overhangs and are produced only antisense to their mRNA templates. To identify canonical siRNAs in C. elegans, we first characterized the siRNAs produced from exogenous dsRNA. As predicted based on earlier studies, exogenous dsRNA is processed into ~23-nt duplexes with 2-4-nt 3'-overhangs, ultimately yielding siRNAs devoid of 5' G-containing sequences that bind with high affinity to the Argonaute RDE-1. Leveraging these characteristics, we searched for their endogenous counterparts and identified thousands of endogenous loci representing dozens of unique elements that give rise to mostly low to moderate levels of siRNAs, called 23H-RNAs. These loci include repetitive elements, alleged coding genes, pseudogenes, non-coding RNAs, and unannotated features, many of which adopt hairpin structures reminiscent of the hpRNA/RNA interference (RNAi) pathway in flies and mice. Our results expand the known repertoire of C. elegans small RNAs and demonstrate that key features of the endogenous siRNA pathway are relatively unchanged in animals. ### Competing Interest Statement The authors have declared no competing interest.