RNA-Seq of different C. elegans gentotype exposed to Heat shock and Recovery
Piwi-interacting RNAs (piRNAs) are best known for silencing transposons and other nonself sequences, yet in Caenorhabditis elegans, they can also target nearly all germline transcripts. The functions of this broad targeting remain unclear. Here, we show that during the heat shock response, piRNAs target the essential heat-shock protein (hsps) mRNAs and other transcripts—normally absent but massively upregulated upon stress—to generate abundant sequence-specific secondary RNA (22G-RNAs) antisense to these mRNAs. Unexpectedly, instead of enforcing silencing, these 22G-RNAs associate with nascent hsp transcripts and RNA polymerase II to delay splicing.  This limits the immediate availability of the newly expressed hsp mRNAs, but preserves a pool of pre-mRNAs for post-transcriptional splicing, ensuring proper maturation and expression of stress-induced and long-intron-containing genes. In piRNA-deficient prde-1 mutants, splicing is precocious, long-intron gene expression is impaired, hsp pre-mRNA is depleted, and transcripts accumulate errors. Consequently, embryos lacking piRNAs become critically dependent on nonsense-mediated decay (NMD) for survival. Thus, by delaying splicing under stress, piRNAs safeguard transcript fidelity and complement NMD as a mechanism to maintain RNA quality control. Our findings reveal a previously unrecognized role for piRNAs in regulating splicing and transcriptome fidelity, linking transposon silencing pathways to RNA quality control.