Restricted Access
Reason: Access restricted by the author. A copy can be requested for private research and study by contacting your institution's library service. This copy cannot be republished
Functional analysis of the TRIM-NHL family protein, NHL-2, in the germline of Caenorhabditis elegans
thesis
posted on 2017-02-22, 03:52 authored by Davis, Gregory MarkTRIM-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.
History
Principal supervisor
Peter BoagYear of Award
2015Department, School or Centre
Biomedical Sciences (Monash Biomedicine Discovery Institute)Additional Institution or Organisation
Biochemistry and Molecular BiologyCampus location
AustraliaCourse
Doctor of PhilosophyDegree Type
DOCTORATEFaculty
Faculty of Medicine Nursing and Health SciencesUsage metrics
Categories
No categories selectedKeywords
Licence
Exports
RefWorks
BibTeX
Ref. manager
Endnote
DataCite
NLM
DC