RG motifs promote piRNA-mediated gene silencing in  <i>C. elegans</i>

Argonaute proteins are essential components of RNA silencing pathways, and their N-terminal extensions, particularly in the PIWI clade, often harbor conserved RG motifs. Despite their prevalence, the function of these motifs remains poorly understood. In this study, we investigate the RG motifs in the N-terminal region of Caenorhabditis elegans PRG-1, a PIWI clade Argonaute. Sequence alignment across Caenorhabditis species reveals three conserved RG motifs, which we confirm to be methylated by mass spectrometry. The region surrounding these motifs is intrinsically disordered, as predicted by disorder algorithms and structural modeling. While the RG motifs are critical for fertility, germline morphology, and Piwi-interacting RNAs (piRNAs) silencing, they are not required for PRG-1 expression, localization, or piRNA loading. Notably, mutation of the RG motifs results in defects in downstream small RNA production, specifically the depletion of WAGO-class small interfering RNAs (siRNAs), without affecting piRNA biogenesis. These findings suggest that the RG motifs of PRG-1 play a crucial role in linking piRNA-mediated silencing to siRNA production, and that their function is critical for fertility and germline maintenance in C. elegans. Despite these defects, the phenotypic severity in the RG mutant is milder than in a PRG-1 null mutant, highlighting the complexity of PRG-1 function and its post-translational modifications.