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

Abstract

SUMMARYThe C. elegans genome encodes nineteen functional Argonaute proteins that utilize 22G-RNAs, 26G-RNAs, miRNAs, or piRNAs to regulate their target transcripts. Only one of these proteins is essential under normal laboratory conditions: CSR-1. While CSR-1 has been studied in various developmental and functional contexts, nearly all studies investigating CSR-1 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 epitopes into the long (CSR-1a) and short (CSR-1b) isoforms of CSR-1, we identified differential expression patterns for the two isoforms. CSR-1a is expressed specifically during spermatogenesis and in select somatic tissues, including the intestine. In contrast, CSR-1b, is expressed constitutively in the germline. Essential functions of csr-1 described in the literature coincide with CSR-1b. In contrast, CSR-1a plays tissue specific functions during spermatogenesis, where it integrates into a spermatogenesis sRNA regulatory network including ALG-3, ALG-4, and WAGO-10 that is necessary for male fertility. CSR-1a is also required in the intestine for the silencing of repetitive transgenes. Sequencing of small RNAs associated with each CSR-1 isoform reveals that CSR-1a engages with 22G- and 26G-RNAs, while CSR-1b interacts with only 22G-RNAs to regulate distinct groups of germline genes and regulate both sperm and oocyte-mediated fertility.

Germ Granules Allow Transmission of Small RNA-Based Parental Responses in the “Germ Plasm”

“…Altogether, these results lead the authors to suggest that CDE-1-dependent uridylation of small RNAs promotes the transition of siRNAs from the CSR-1 argonaute to WAGO-4 to facilitate their inheritance ( Xu et al., 2018 ). The recent finding that like WAGO-4, the CSR-1b argonaute localizes to the Z germ granule is in accordance with this view ( Charlesworth et al., 2020 ; Wan et al., 2018 ). The argonaute HRDE-1 is crucial for RNAi inheritance ( Ashe et al., 2012 ; Buckley et al., 2012 ; Kalinava et al., 2017 ; Lev et al., 2017 ; Luteijn et al., 2012 ; Pak et al., 2012 ; Shirayama et al., 2012 ).…”

“…Untemplated poly-urydilation of siRNAs is thought to be involved in this process. The siRNAs that bind with the CSR-1 argonaute (specifically the CSR-1b isoform, Charlesworth et al., 2020 ; van Wolfswinkel et al., 2009 ), tend to have longer untemplated poly-U tails when compared with the siRNAs that bind with the WAGO-1 and HRDE-1 argonauts ( de Albuquerque et al., 2015 ; Houri-Ze'evi et al., 2016 ; Lev et al., 2019b ; Phillips et al., 2015 ; van Wolfswinkel et al., 2009 ). Poly-uridylation of siRNAs is partially dependent on CDE-1, a germ granule-localized poly uracil polymerase ( van Wolfswinkel et al., 2009 ; Xu et al., 2018 ).…”

Small RNAs and chromatin in the multigenerational epigenetic landscape ofCaenorhabditis elegans

“…Because of this, it has been suggested that these two pathways compete for binding of the same cohort of 22Gs to control the expression of germline genes [13]. Interestingly, these proteins comprise two of the only three components currently shown to localize to the newly identified Z-granules [61,62] (see below). This overlap could provide an explanation for the silencing observed against some of the CSR-1-22G-targeted genes; possibly the CSR-1-class 22Gs that cause silencing are bound not by CSR-1, as their name suggests, but instead by WAGO-4.…”

“…Many small RNA pathway proteins localize to P granules, including Dicer, PRG-1 and CSR-1 [38,50,74,75]. The functions of these proteins are heavily intertwined with correct P granule formation: these proteins depend on P granule compartmentation to carry out their functions and P granules depend on these proteins for their proper structure and localization [62,74,75].…”

Nematode Small RNA Pathways in the Absence of piRNAs

“…AsCSR-1 is not expressed in late embryos, larvae or somatic tissues and the gene becomes heavily marked with H3K9me3 in 32-64 cell embryos when its expression ceases (Figure S1). Two isoforms of CSR-1 protein are present in C. elegans that vary in the first exon and thus amino-terminus [62][63][64][65]. The C. elegans CSR-1a isoform with the extended aminoterminus is expressed during spermatogenesis [64].…”