February 18, 2025

b Western blot detecting CSR-1 expression, using -CSR-1 antibodies, in wild-type animals at L4 stage

b Western blot detecting CSR-1 expression, using -CSR-1 antibodies, in wild-type animals at L4 stage. the germline and during all stages of development while CSR-1A expression is restricted to germ cells undergoing spermatogenesis. Furthermore, CSR-1A associates preferentially with 22G-RNAs mapping to spermatogenesis-specific genes whereas CSR-1B-bound small RNAs map predominantly to oogenesis-specific genes. Interestingly, the exon unique to CSR-1A contains multiple dimethylarginine modifications, which RU 24969 hemisuccinate are necessary for the preferential binding of CSR-1A to spermatogenesis-specific 22G-RNAs. Thus, we have discovered a regulatory mechanism for Argonaute proteins that allows for specificity of small RNA binding between similar Argonaute proteins with overlapping temporal and spatial localization. germ cells promote this selective environment is by way of a perinuclear structure called the P granule4. These germline granules, which help to ensure pluripotency and germ cell identity, form a phase-separated condensate outside of the nucleus and contiguous with nuclear pores, where they regulate newly synthesized germline transcripts4C7. One key regulator of P granule morphology and germline development is the Argonaute protein, CSR-1 (Chromosome Segregation and RNAi deficient)8,9. Argonaute proteins are the core effectors of all small RNA-related pathways. These proteins bind small guide RNAs and regulate complementary transcripts by RU 24969 hemisuccinate either directly cleaving target RNAs or recruiting cofactors that promote transcriptional or posttranscriptional gene regulation10,11. Of the ~27 Argonaute proteins annotated in the genome, only CSR-1 is essential for fertility, and it is reported to regulate over 4000 germline-expressed genes8,12. CSR-1 binds to a class of antisense 22-nucleotide small interfering RNAs with a 5 guanosine (22G-RNAs) whose target transcripts are thought to be protected from silencing by other small RNA-mediated pathways, and are thus licensed for germline expression8,13,14. In contrast, foreign DNA such as transposons and transgenes containing non-sequences are recognized by piwi-interacting RNAs (piRNAs, also known as 21U-RNAs in mutant embryos, chromosomes fail to properly align on the metaphase plate, resulting in aberrant mitotic division and anaphase bridging8,12,20. In the adult germline, loss of CSR-1 leads to fewer germ cells, defects in meiotic progression, and a delayed spermatogenesis to oogenesis switch21. Additionally, CSR-1 has been implicated in a myriad of other cellular processes, including maturation of core histone mRNAs, promotion of sense-oriented RNA RU 24969 hemisuccinate polymerase II transcription, attenuation of translation elongation, prevention of premature activation of embryonic transcripts in oocytes, alternative splicing, and paternal inheritance22C27. Yet how one Argonaute protein can regulate so many seemingly distinct processes remains unanswered. Here we demonstrate that CSR-1 has two isoformsCSR-1B, which is present throughout the germline and CSR-1A, which is specific to spermatogenic germ cells. CSR-1A and CSR-1B associate with distinct subsets of small RNAs to regulate spermatogenic or oogenic transcripts, respectively. The specificity of the two CSR-1 isoforms is interesting, considering they share nearly complete sequence homology and co-localize at the P granule in L4 larval and male germlines. We found that HLA-DRA the first exon of CSR-1A is modified at arginine/glycine (RG) motifs by dimethylarginine. Here we show that loss of the dimethylarginine results in the loss of CSR-1A specificity for its preferred spermatogenic small RNA partners, resulting in CSR-1A indiscriminately binding to both spermatogenic and oogenic siRNAs. Thus, in this study, we have identified the first instance of methylarginine modification of a Argonaute protein and demonstrated a previously unappreciated mechanism by which Argonaute proteins can acquire small RNA specificity. Results The long isoform of CSR-1 is selectively expressed during spermatogenesis The Argonaute protein, CSR-1, has two isoforms, though previously little was known about their RU 24969 hemisuccinate distinct functions. The longer isoform, referred to as CSR-1A, and the shorter isoform, CSR-1B, share complete sequence homology, except for a unique exon at the 5 end of CSR-1A (Fig.?1a). Both isoforms are expressed, though at differential levels (Fig.?1b)8,20. Furthermore, transcriptome analysis suggests that CSR-1A is expressed in spermatogenic gonads, but excluded from oogenic gonads28. To explore the distinct functions of the two CSR-1 isoforms, we used CRISPR/Cas9 to endogenously tag CSR-1A at its N-terminus with a 2xHA/mCherry tag. We also tagged both CSR-1A and CSR-1B in the same strain by adding a 3xFLAG/GFP tag to the N-terminus of CSR-1B (Fig.?1a). When both isoforms are tagged, CSR-1A?+?B is strongly expressed.