Biogenesis of C. elegans spermatogenesis small RNAs is initiated by a zc3h12a like ribonuclease

<p><span>Small RNAs regulate spermatogenesis in many species, ranging from <em>Caenorhabditis elegans</em> to mammals. In <em>C. elegans</em>, two Argonaute proteins, ALG-3 and ALG-4, and their associated <em>alg-3/4</em> 26G-small RNAs are essential for spermatogenesis at 25°C and for imprinting paternal memory of germline gene expression in offspring. The <em>alg-3/4</em> 26G-small RNAs are antisense to their target mRNAs and are known to be produced by </span><span>the</span><span> RNA-dependent RNA polymerase, RRF-3. However, it remains unclear how the RNA templates for RRF-3 are generated and which cellular processes are affected by </span><em><span>alg-3/4</span></em><span> 26G-small RNAs</span><span>. Here, we demonstrate a key role for the conserved zc3h12a-ribonuclease-like NYN-domain-containing protein, NYN-3, in spermatogenesis at 25°C. Expression of NYN-3 was temporally coordinated with ALG-3, and our sequencing of both total and 2xFLAG::ALG-3-immunoprecipitated small RNAs revealed that NYN-3 is required for the biogenesis of <em>alg-3/4</em> 26G small RNAs. We further used ePAR-CLIP sequencing to identify NYN-3 binding sites on <em>alg-3/4</em>-targeted mature mRNAs; the NYN-3 binding sites were downstream of the binding sites for <em>alg-3/4</em> 26G-small RNAs. Additionally, 3'-RACE results placed the NYN-3 mRNA recognition upstream of RRF-3 binding and further revealed NYN-3 cleavage sites in <em>alg-3/4</em>-targeted mRNAs. Finally, a bioinformatics analysis was performed to parse the 26G small RNA-targeted genes into functional subclasses (e.g., signaling, chromatin defects). Collectively, these findings reveal NYN-3 as an initiator of small RNA generation that is central to the coordination of 26G small RNA-mediated gene regulation during spermatogenesis.</span></p>