In every domain of life, Argonaute proteins and their associated small RNAs were found to regulate gene expression. In the eumetazoan germline, Piwi clade Argonautes associate with piRNAs to control transposable elements. Given the potentially detrimental role of transposon expression, Piwi activity has a pivotal role in maintaining fertility. The conserved CHHC zinc-finger protein gametocyte-specific factor 1 (Gtsf1), as well as its paralogs Gtsf1l and Gtsf2, interact with Piwi proteins in flies and mice. In Drosophila, Gtsf1 interacts with Piwi to elicit transcriptional gene silencing. However, mechanistic insights into Gtsf1 function are lacking. In Caenorhabditis elegans, Piwi is required for transgenerational fertility while Argonautes and the RNA-dependent RNA Polymerase (RdRP) RRF-3 of the 26G-RNA pathway are required for fertility at elevated temperatures. We identified T06A10.3 as the C. elegans Gtsf1 ortholog and named it
gtsf-1. Given its importance in Piwi-mediated transcriptional silencing in D. melanogaster, we addressed the role of
gtsf-1 in the context of the small RNA pathways of C. elegans. To this purpose, we used CRISPR-Cas9 technology to produce mutant alleles of
gtsf-1. Surprisingly, using reporter strains, we found that
gtsf-1 is not involved in the piRNA pathway and transposon silencing in C. elegans. Instead,
gtsf-1 mutant animals recapitulate the mutant phenotypes of factors operating in the 26G-RNA pathway, such as those of RRF-3. These mutant phenotypes include, notably, temperature-sensitive sterility at 25oC. Accordingly, small RNA sequencing showed that 26G-RNAs are severely depleted in
gtsf-1 mutants. Strikingly, by immunoprecipitation of GTSF-1 followed by quantitative proteomics, we identified RRF-3 as a strong interactor of GTSF-1. Furthermore, GTSF-1 interacts with RRF-3 via its two CHHC zinc-fingers. In embryos, where a functional RRF-3 associated small RNA-producing complex is present, GTSF-1 is required for complex assembly. We propose that GTSF-1 homologs in other species may similarly act to drive the assembly of larger complexes that subsequently act in small RNA production or in imposing small RNA-mediated silencing activities.