Endogenous small RNA pathways play vital roles in regulating gene expression to ensure germline integrity across animal species. The key effectors of these pathways are Argonaute proteins (AGOs), which utilize small RNA partners to identify target transcripts in a sequence specific manner. Although AGOs predominantly downregulate their target transcripts, the essential C. elegans AGO CSR-1 has been shown to promote and fine tune germline gene expression. Remarkably, although CSR-1 is relatively well studied among C. elegans AGOs, one key feature that has been overlooked is the functional distinction between two isoforms of the protein. These two isoforms differ by 163 residues at the N terminus, in a region enriched in RG motifs (longer isoform: CSR-1a; shorter isoform: CSR-1b). In other AGOs, RG motifs are methylated, which impacts the recruitment of key AGO co-factors, such as Tudor proteins. We hypothesize that the two isoforms of CSR- have distinct functional roles in development. Using a series of
csr-1 transgenes (MosSci), endogenously-tagged
csr-1 strains (CRISPR/Cas9), and two different antisera that recognize 1) both isoforms of CSR-1 or 2) CSR-1a only, we have identified the key differences between the CSR-1 isoforms. First, the CSR-1 isoforms have distinct expression profiles-CSR-1a is expressed in the germline only during spermatogenesis, while CSR-1b is constitutively expressed in the germline. Second, complementation and phenotypic assays point to CSR-1b as the essential isoform associated with previously described
csr-1 phenotypes, while CSR-1a is required for wild-type fertility at high temperatures (Mortal Germline phenotype). Third, CSR-1a and CSR-1b bind to different classes of small RNAs (CSR-1a: 26G-RNAs versus CSR-1b: 22G-RNAs). Overall, our data point to a critical and previously unappreciated role for CSR-1a in spermatogenesis, which it may execute in concert with ALG-3, -4, and another uncharacterized WAGO that are also expressed specifically during spermatogenesis. Our current studies are aimed and understanding how CSR-1a regulates its mRNA targets, and identifying the partners of each CSR-1 isoform.