RNA silencing is a critically important mechanism through which cells regulate gene expression and protect the genome against aberrant RNAs, transposons, and viruses. This suppression of aberrant transcripts is carried out by the evolutionarily conserved small RNA pathway. Small RNAs are loaded onto Argonaute proteins to induce silencing through sequence recognition of specific transcripts through a variety of different silencing mechanisms including post-transcriptional or co-transcriptional silencing. In recent studies, it has been shown that some Argonaute proteins are modified with dimethylarginine within arginine/glycine-rich regions (RG/RGG motifs). In D. melanogaster, dimethylation of the Argonaute protein Aub is required for assembly of the ping-pong complex and ultimately for piRNA mediated transposon silencing. However, it remains unclear whether there are any general principles underlying the function of Argonaute modification by dimethylation. First, to determine if any of the 27 C. elegans Argonaute proteins are dimethylated, we identified Argonaute proteins containing RG/RGG motifs. The proteins with the highest incidence of these motifs are the cytoplasmic Argonautes ERGO-1, CSR-1, ALG-3 and PRG-1, and the nuclear Argonaute HRDE-1. These five Argonaute proteins were tagged using CRISPR and immunoprecipitated. Following mass spectrometry analysis, we identified multiple dimethylation sites within each of these Argonaute proteins. Next, to understand the physiological role of these dimethylarginine modifications, we used CRISPR to generate methylation-defective mutant Argonaute proteins. We are currently focusing on the PRG-1 RG-motif mutant, and assessing how mRNA and small RNA expression change compared to wild-type and a
prg-1 null mutant. To further probe PRG-1 function in the absence of dimethylarginine, we will next determine whether the PRG-1 RG-motif mutant activates a sensor for piRNA silencing activity and whether it has transgenerational fertility defects. This work will define the functional relevance of Argonaute protein methylation in the C. elegans germline.