Pachytene piRNAs control discrete meiotic events during spermatogenesis and restrict gene expression in space and time

Pachytene piRNAs, a Piwi-interacting RNA subclass in mammals, are hypothesized to regulate non-transposon sequences during spermatogenesis. <i>Caenorhabditis elegans</i> piRNAs, the 21URNAs, are implicated in regulating coding sequences; the messenger RNA targets and biological processes they control during spermatogenesis are largely unknown. We demonstrate that loss of 21URNAs compromises homolog pairing and makes it permissive for nonhomologous synapsis resulting in defects in crossover formation and chromosome segregation during spermatogenesis. We identify Polo-like kinase 3 (PLK-3), among others, as a 21URNA target. 21URNA activity restricts PLK-3 protein to proliferative cells, and expansion of PLK-3 in pachytene overlaps with the meiotic defects. Removal of <i>plk-3</i> results in quantitative genetic suppression of the meiotic defects. One discrete 21URNA inhibits PLK-3 expression in late pachytene cells. Together, these results suggest that the 21URNAs function as pachytene piRNAs during <i>C. elegans</i> spermatogenesis. We identify their targets and meiotic events and highlight the remarkable intricacy of this multi-effector mechanism during spermatogenesis.