A low-abundance class of Dicer-dependent siRNAs produced from a variety of features in C. elegans
Canonical small interfering RNAs (siRNAs) are generated by the cleavage of double-stranded RNA (dsRNA) by the ribonuclease Dicer. siRNAs are found in plants, animals, and some fungi where they bind to Argonautes to direct RNA silencing. In this study, we characterized the canonical Dicer-dependent siRNAs of C. elegans. We identified thousands of endogenous loci, representing dozens of unique elements, that give rise to 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. Using protein-small RNA co-immunoprecipitation, we identified the Argonautes that associate with 23H-RNAs and using mRNA-sequencing we explored their roles in gene regulation. Overall design: mRNA and small RNA high-throughput sequencing were done on wild-type and rde-1(ram40) mutant strains of C. elegans, along with small RNA sequencing from various transgenic strains containing GFP fusions to Argonaute proteins. Samples were either untreated or subjected to RNAi targeting oma-1, nrfl-1, dcr-1, or rde-1. RNA was isolated from cell lysates or Argonaute co-immunoprecipitates from C. elegans cultured at 20°C. mRNA sequencing data was analyzed with a custom pipeline utilizing fastp, RSEM, STAR, and DESeq2. Small RNA data was analyzed with the tinyRNA pipeline complemented by custom Python scripts.