Transcriptome-wide analyses of piRNA binding sites suggest distinct mechanisms regulate piRNA binding and silencing in C. elegans .

PIWI-interacting RNAs (piRNAs) protect genome integrity by silencing transposon mRNAs and some endogenous mRNAs in various animals. However, piRNAs only trigger gene silencing at select predicted targeting sites, suggesting additional cellular mechanisms regulate piRNA silencing. To gain insight into possible mechanisms, we compared the transcriptome-wide predicted piRNA targeting sites to the in vivo piRNA binding sites. Surprisingly, while sequence-based predicted piRNA targeting sites are enriched in 3' UTRs, we found that piRNAs preferentially bind to coding regions (CDS) of target mRNAs, leading to preferential production of secondary silencing small RNAs in the CDS. However, our analyses suggest that this CDS binding preference cannot be explained by the action of antisilencing Argonaute CSR-1. Instead, our analyses imply that CSR-1 protects mRNAs from piRNA silencing through two distinct mechanisms-by inhibiting piRNA binding across the entire CSR-1 targeted transcript, and by inhibiting secondary silencing small RNA production locally at CSR-1 bound sites. Together, our work identifies the CDS as the critical region that is uniquely competent for piRNA binding in We speculate the CDS binding preference may have evolved to allow the piRNA pathway to maintain robust recognition of RNA targets in spite of genetic drift. Together, our analyses revealed that distinct mechanisms are responsible for restricting piRNA binding and silencing to achieve proper transcriptome surveillance.