piRNAs safeguard splicing and RNA fidelity

Piwi-interacting RNAs (piRNAs) silence transposons and foreign sequences; yet, for poorly understood reasons, they also extensively target endogenous mRNAs. Here, we demonstrate that this targeting preserves transcript isoform fidelity by regulating pre-mRNA splicing in C. elegans. Using the heat shock response to provoke rapid transcriptional changes, we found that piRNAs and PIWI/PRG-1 direct the production of endogenous antisense 22G-RNAs from newly induced transcripts. These endo-siRNAs associate with RNA Polymerase II, spliceosome protein MOG-7/C2orf3/Ntr2, and nascent RNAs to delay splice completion and regulate splice-site selection. Long-read transcriptome sequencing revealed that loss of piRNAs accelerates splice completion but compromises accurate splice-site selection, increasing the production of aberrant transcript isoforms that are normally eliminated by alternative splicing coupled to nonsense-mediated decay (AS-NMD). Consequently, piRNA pathway mutants are profoundly reliant on NMD for embryonic survival, development, and proteostasis maintenance. Together, our findings identify a previously unrecognized role for the piRNA pathway in coordinating splicing with RNA quality control to preserve proteome integrity. We propose that this unexpected role for the piRNA pathway may have evolved from its ancestral function in recognizing transposable elements, which frequently introduce cryptic splice sites and undergo exonization, to generate novel transcripts that require elimination by NMD.