Pre-piRNA trimming safeguards piRNAs against erroneous targeting by RNA-Dependent RNA Polymerase

In animal germ lines, The Piwi/piRNA pathway plays a crucial role in safeguarding genome integrity and promoting fertility. Following transcription from discrete genomic loci, piRNA precursors undergo nucleolytic processing at both 5’ and 3’ ends. The ribonuclease PARN-1 and its orthologs mediate piRNA 3' trimming in worms, insects and mammals. Yet, the significance of this evolutionarily conserved processing step is not well understood. Employing C. elegans as a model organism, our recent work has demonstrated that 3' trimming protects piRNAs against non-templated nucleotide additions and degradation. In this study, we present an unexpected finding that C. elegans deficient for PARN-1 accumulate a heretofore uncharacterized RNA species termed anti-piRNAs, which are antisense to piRNAs. These anti-piRNAs associate with Piwi proteins and display the propensity for a length of 17-19 nucleotides and 5’ guanine and adenine residues. We show that untrimmed pre-piRNAs in parn-1 mutants are modified by the terminal nucleotidyl transferase RDE-3 and erroneously targeted by the RNA dependent RNA polymerase EGO-1, thereby giving rise to anti-piRNAs. Taken together, our work identifies a previously unknown class of small RNAs upon loss of parn-1 and provides mechanistic insight to activities of RDE-3, EGO-1 and Piwi proteins. Analysis of small RNAs from wild-type, parn-1 and various parn-1 double mutants