Two isoforms of the essential C. elegans Argonaute CSR-1 differentially regulate sperm and oocyte fertility

Abstract

The Caenorhabditis elegans genome encodes nineteen functional Argonaute proteins that use 22G-RNAs, 26G-RNAs, miRNAs or piRNAs to regulate target transcripts. Only one Argonaute is essential under normal laboratory conditions: CSR-1. While CSR-1 has been studied widely, nearly all studies have overlooked the fact that the csr-1 locus encodes two isoforms. These isoforms differ by an additional 163 amino acids present in the N-terminus of CSR-1a. Using CRISPR-Cas9 genome editing to introduce GFP::3xFLAG into the long (CSR-1a) and short (CSR-1b) isoforms, we found that CSR-1a is expressed during spermatogenesis and in several somatic tissues, including the intestine. CSR-1b is expressed constitutively in the germline. small RNA sequencing of CSR-1 complexes shows that they interact with partly overlapping sets of 22G-RNAs. Phenotypic analyses reveal that the essential functions of csr-1 described in the literature coincide with CSR-1b, while CSR-1a plays tissue specific functions. During spermatogenesis, CSR-1a integrates into an sRNA regulatory network including ALG-3, ALG-4 and WAGO-10 that is necessary for fertility at 25°C. In the intestine, CSR-1a silences immunity and pathogen-responsive genes, and its loss results in improved survival from the pathogen Pseudomonas aeruginosa. Our findings functionally distinguish the CSR-1 isoforms and highlight the importance of studying each AGO isoform independently.

piRNAs initiate transcriptional silencing of spermatogenic genes during C. elegans germline development

“…We combined our worm population sorting strategy with CSR-1 or HRDE-1 IPs, followed by small RNA sequencing. Given the expression of two isoforms of CSR-1 in the L4 stage ( Charlesworth et al., 2021 ; Nguyen and Phillips, 2021 ), we verified the enrichment of both CSR-1 isoforms 22G-RNA targets in our sorted CSR-1 IPs ( Figure S7 A). The analysis of 22G-RNAs from sorted early L4 to young adult worms showed that oogenic 22G-RNAs were globally loaded into CSR-1 but not in HRDE-1 ( Figure S7 B).…”

“…We followed the subcellular localization of piRNA pathway components during spermatogenesis to discern the spatiotemporal specificity of nuclear piRNA-dependent signaling. In parallel, we studied the localization of CSR-1, a germline AGO protein highly enriched in P granules ( Claycomb et al., 2009 ) and also present in Z granules ( Charlesworth et al., 2021 ), another type of biomolecular condensate part of the C. elegans nuage ( Wan et al., 2018 ). We confirmed the co-localization of PIWI and CSR-1 with GLH-1, the C. elegans homolog of the DEAD-box RNA helicase Vasa, a core P granule component ( Figure S2 A).…”

“…The fact that HRDE-1 is not the only AGO loading 22G-RNAs and targeting spermatogenic genes might explain why the absence of piRNAs does not cause a global loss of spermatogenic 22G-RNAs. Indeed, the CSR-1 pathway also targets spermatogenic mRNAs ( Charlesworth et al., 2021 ; Nguyen and Phillips, 2021 ). Thus, only by filtering 22G-RNA populations loaded into HRDE-1 in the presence or absence of piRNAs we were able to find a specific subset of 22G-RNAs following the established criteria to define putative direct piRNA regulation.…”

CSR-1 RNA interference pathway restricts holocentromere protein CENP-A/HCP-3 localization in Caenorhabditis elegans

“…CSR-1 suppresses HCP-3 via its RNAi pathway and with its slicing activity. The regulation likely happens in the hermaphrodite germline where the CSR-1 isoform b is enriched (Charlesworth et al, 2021). Specific depletion of the other soma cells-enriched isoform a does not cause embryonic lethality (Claycomb et al, 2009, Gerson-Gurwitz et al, 2016, chromosome missegregation, as well as a dramatic GFP::HCP-3 elevation as the depletion of both isoforms does.…”

“…To deplete only CSR-1A, we use dsRNA targeting the first exon of isoform A. As depletion of CSR-1A alone is not embryonic lethal (Gerson-Gurwitz et al, 2016, Charlesworth et al, 2021, we validated the csr-1a knockdown by RT-PCR, whose mRNA level is 12.23% of that in the untreated embryos (Figure S3B). Depletion of CSR-1A results in 1.3-fold increase in chromatin GFP::HCP-3 level in 1-cell stage embryos, compared to the untreated control (Figure 3E).…”