Maternal mRNA regulation is essential to germline and embryo development in metazoans. Over the past few decades, it has become clear that many RNA-binding proteins (RBPs) containing highly conserved RNA-binding domains orchestrate spatiotemporal expression pattern of germline and embryonic genes to control gametogenesis and embryogenesis in the nematode Caenorhabditis elegans. These RBPs bind regulatory elements situated primarily in the UTRs of their target mRNAs to regulate expression by influencing transcript stability or translational efficiency. The 3´UTR is the main determinant of patterned expression in the germline of C. elegans. MEX-3 is a KH-domain RBP that is required for anterior cell fate specification and maintenance of germ cell totipotency. MEX-3 is expressed in mitotic germ cells, maturing oocytes, and early embryos. MEX-3 is absent in the meiotic pachytene region as well as the diplotene loop region. The 3´UTR of mex-3 is sufficient to confer MEX-3’s expression to a transgenic reporter. Here, I assessed the importance of the endogenous 3´UTR of mex-3 to MEX-3’s expression pattern and function using CRISPR/Cas9 mutagenesis followed by molecular and phenotypic analysis. 3´UTR deletion allelic series demonstrated that the endogenous 3´UTR of mex-3 is indeed required for MEX-3’s pattern in the germline in vivo. I identified regions of the 3´UTR that contribute to repression of MEX-3 in different regions of the germline. Surprisingly, the 3´UTR was dispensable for viability. However, several 3´UTR deletions exhibited reduced fertility. Analysis of the transcriptome of these mutants revealed that the 3´UTR deletions altered expression of soma-specific genes, consistent with MEX-3’s role in repressing somatic gene programs. These data sets also showed that mex-3 mRNA levels do not correlate with MEX-3 protein levels.
In order to determine which germline RBPs regulate expression of mex-3 through its 3´UTR, I used RNAi to knock down several candidate RBPs including three that were previously shown to regulate expression of MEX-3. My RNAi studies showed that GLD-1, LIN-41, and OMA-1/2 repress expression of mex-3 through its 3´UTR in the meiotic pachytene region, diplotene loop region, and oocytes in the proximal end, respectively. Furthermore, I have identified DAZ-1, an RRM-containing RBP, as a novel repressor of MEX-3 expression in the distal mitotic germ cells. Using RNAi, I demonstrated that poly(A) tail length control and the translation initiation factor IFE-3 contribute to MEX-3’s expression in the germline. Poly(A) polyadenylation and deadenylation cycles govern expression of mex-3 in the distal mitotic germ cells, while IFE-3 contributes to repression of mex-3 in the meiotic pachytene region, presumably by control of translation initiation. Using high throughput sequencing-based poly(A) tail assay, I have shown that the poly(A) tail length distribution of mex-3 mRNA shifts towards shorter tails in the mex-3 3´UTR deletion mutants with reduced fertility phenotypes. Our study is the first as far as we know to address the importance of an endogenous 3´UTR to in vivo expression and function in C. elegans germline. It will be interesting to determine how different RBPs and cis-regulatory elements orchestrate the spatiotemporal expression pattern of a single germline gene. It will also be interesting to assess whether other germline 3´UTRs are similarly dispensable for viability, and if so, what role do 3´UTRs play in enhancing reproductive success.
| Identifer | oai:union.ndltd.org:umassmed.edu/oai:escholarship.umassmed.edu:gsbs_diss-2166 |
| Date | 09 September 2021 |
| Creators | Albarqi, Mennatallah M.Y. |
| Publisher | eScholarship@UMMS |
| Source Sets | University of Massachusetts Medical School |
| Detected Language | English |
| Type | text |
| Format | application/pdf |
| Source | GSBS Dissertations and Theses |
| Rights | Copyright is held by the author, with all rights reserved., select |
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