Systematic characterization of small RNAs associated with C. elegans Argonautes

Argonaute proteins generally play regulatory roles by forming complexes with the corresponding small RNAs (sRNAs). An expanded Argonaute family with 20 potentially functional members has been identified in Caenorhabditis elegans. Canonical sRNAs in C. elegans are miRNAs, small interfering RNAs including 22G-RNAs and 26G-RNAs, and 21U-RNAs, which are C. elegans piRNAs. Previous studies have only covered some of these Argonautes for their sRNA partners, and thus, a systematic study is needed to reveal the comprehensive regulatory networks formed by C. elegans Argonautes and their associated sRNAs. We obtained in situ knockin (KI) strains of all C. elegans Argonautes with fusion tags by CRISPR/Cas9 technology. RNA immunoprecipitation against these endogenously expressed Argonautes and high-throughput sequencing acquired the sRNA profiles of individual Argonautes. The sRNA partners for each Argonaute were then analyzed. We found that there were 10 Argonautes enriched miRNAs, 17 Argonautes bound to 22G-RNAs, 8 Argonautes bound to 26G-RNAs, and 1 Argonaute PRG-1 bound to piRNAs. Uridylated 22G-RNAs were bound by four Argonautes HRDE-1, WAGO-4, CSR-1, and PPW-2. We found that all four Argonautes played a role in transgenerational epigenetic inheritance. Regulatory roles of the corresponding Argonaute-sRNA complex in managing levels of long transcripts and interspecies regulation were also demonstrated. In this study, we portrayed the sRNAs bound to each functional Argonaute in C. elegans. Bioinformatics analyses together with experimental investigations provided perceptions in the overall view of the regulatory network formed by C. elegans Argonautes and sRNAs. The sRNA profiles bound to individual Argonautes reported here will be valuable resources for further studies.