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Functional analysis of the TRIM-NHL family protein, NHL-2, in the germline of Caenorhabditis elegans
thesisposted on 2017-02-22, 03:52 authored by Davis, Gregory Mark
TRIM-NHL family proteins are highly conserved and regulate various cellular processes by functioning as co-factors of the micro RNA pathway (miRNA), or as E3 ubiquitin ligases. A member of this family, NHL-2, has previously been shown to function as a miRNA co-factor in the somatic tissues of Caenorhabditis elegans. Additionally, NHL-2 is highly expressed in germ cells, although its function in this tissue is unknown. Therefore, this study used a combination of genome-wide RNAi screening, deep-sequencing and proteomics to explore the role of NHL-2 and its contribution to germline function. Genome-wide RNAi screening identified 42 genes that gave strong synthetic phenotypes when knocked down in nhl-2 null mutants. This included members of the WAGO and CSR-1 small 22G RNA pathways, including drh-3, ekl-1 and cde-1. Knockdown of these genes in nhl-2 null mutants leads to abnormal chromosomal morphology, reduced brood size and highly penetrant embryonic lethality. In addition to this, nhl-2 is required for the downregulation of WAGO-1 22G RNA targets and deep-sequencing of nhl-2 null mutants shows decreased levels of CSR-1 and WAGO-1 small 22G RNAs. Levels of miRNAs and 21U RNAs appear unchanged in nhl-2 null mutants, suggesting that nhl-2 is specifically required for 22G RNA biogenesis or stability. Furthermore, NHL-2 associates with CSR-1 as well as the ubiquitin machinery, but absence of nhl-2 does not alter CSR-1 levels, suggesting that this interaction is not associated with CSR-1 turnover. Collectively, this study suggests that NHL-2 maintains germline function by functioning as a co-factor of the WAGO and CSR-1 small RNA pathways.