Maternally supplied mRNAs encode for necessary developmental regulators that pattern early embryos in many species until zygotic transcription is activated. In Caenorhabditis elegans, post-transcriptional regulatory mechanisms guide early development during embryogenesis. Maternal transcripts remain in a translationally silenced state until fertilization. A suite of RNA-binding proteins (RBP’s) regulate these maternally supplied mRNAs during oogenesis, the oocyte-to-embryo transition, and early embryogenesis. Identifying the target specificity of these RNA-binding proteins will reveal their contribution to patterning of the embryo. We are studying post-transcriptional regulation of maternal mRNAs during oocyte maturation, which is an essential part of meiosis that prepares oocytes for fertilization. Although the physiological events taking place during oocyte maturation have been well studied, the molecular mechanisms that regulate oocyte maturation are not well understood.
OMA-1 and OMA-2 are essential CCCH-type tandem zinc finger (TZF) RBP’s that function redundantly during oocyte maturation. This dissertation shows that I defined the RNA-binding specificity of OMA-1, and demonstrated that OMA-1/2 are required to repress the expression of 3ʹUTR reporters in developing oocytes. The recovered sequences from in vitro selection demonstrated that OMA-1 binds UAA and UAU repeats in a cooperative fashion. Interestingly, OMA-1 binds with high affinity to a conserved region of the glp-1 3ʹUTR that is rich in UAA and UAU repeats. Multiple RNA-binding proteins regulate translation of GLP-1 protein, a homolog of Notch receptor. In addition to previously identified RBP’s, we showed that OMA-1 and OMA-2 repress glp-1 reporter expression in C. elegans oocytes.
Mapping the OMA-1 dependent regulatory sites in the glp-1 mRNA and characterizing the interplay between OMA-1 and other factors will help reveal how multiple regulatory signals coordinate the transition from oocyte to embryo but the abundance of OMA-1 binding motifs within the glp-1 3ʹUTR makes it infeasible to identify sites with a functional consequence. I therefore first developed a strategy that allowed us to generate transgenic strains efficiently using a library adaptation of MosSCI transgenesis in combination with rapid RNAi screening to identify RBP-mRNA interactions with a functional consequence. This allowed me to identify five novel mRNA targets of OMA-1 with an in vivo regulatory connection. In conclusion, the findings in this dissertation provide new insights into OMA-1 mediated mRNA regulation and provide new tools for C. elegans transgenesis. Development of library MosSCI will advance functional mapping of OMA-1 dependent regulatory sites in the target mRNAs. Extending this strategy to map functional interactions between mRNA targets and RNAbinding proteins in will help reveal how multiple regulatory binding events coordinate complex cellular events such as oocyte to embryo transition and cell-fate specification.
| Identifer | oai:union.ndltd.org:umassmed.edu/oai:escholarship.umassmed.edu:gsbs_diss-1850 |
| Date | 28 April 2016 |
| Creators | Kaymak, Ebru |
| Publisher | eScholarship@UMassChan |
| Source Sets | University of Massachusetts Medical School |
| Detected Language | English |
| Type | text |
| Format | application/pdf |
| Source | Morningside Graduate School of Biomedical Sciences Dissertations and Theses |
| Rights | Copyright is held by the author, with all rights reserved. |
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