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Elucidating Post-transcriptional Regulation of the Vertebrate Segmentation Clock and the Role of Pnrc2-dependent mRNA Decay During Zebrafish Embryogenesis

Blatnik, Monica Crystal
http://rave.ohiolink.edu/etdc/view?acc_num=osu1681929063282294

Abstract Details

2023, Doctor of Philosophy, Ohio State University, Molecular, Cellular and Developmental Biology.
During early vertebrate embryogenesis, muscle and skeletal stem cells are grouped into reiterated segments, called somites, in a process called somitogenesis. Somite formation is established by a genetic oscillator called the segmentation clock, comprised of a network of genes expressed periodically in the presomitic mesoderm. Precise control of segmentation clock oscillations is driven by robust temporal regulation of mRNA production, translation, and mRNA decay, and my work explores post-transcriptional mechanisms that regulate oscillatory expression. proline-rich nuclear receptor coactivator 2 (pnrc2) regulates oscillatory mRNA decay in zebrafish embryos and loss of pnrc2 results in stabilization and accumulation of segmentation clock transcripts. Despite an increase in segmentation clock mRNA abundance, pnrc2 mutant embryos exhibit normal segmentation clock protein expression and somite patterning is not disrupted. I discovered a closely related gene, pnrc1, is upregulated in MZpnrc2-/- mutants and can rescue the her1 misexpression phenotype when overexpressed in pnrc2 mutant embryos. I hypothesized that pnrc1 may partially compensate for loss of function of pnrc2, resulting in normal somitogenesis in MZpnrc2-/- embryos. However, I found that pnrc1-/-;pnrc2-/-double mutant embryos are morphologically indistinguishable from wild-type embryos and are molecularly indistinguishable from pnrc2-/- single mutants, with respect to her1 expression (Chapter 2). To determine the translation status of accumulated transcripts, I performed polysome profiling analysis on wild-type and MZpnrc2-/- mutant embryos at mid-segmentation stage. This revealed segmentation clock gene transcripts that are upregulated upon loss of pnrc2, including her1, her7, and rhov, are significantly increased in the ribosome-unbound (non-translating) fractions, indicating that accumulated transcripts are not efficiently translated, thus explaining the lack of significant protein accumulation (Chapter 2). To identify the landscape of genes affected by loss of pnrc2 function, we conducted RNA-seq analysis on wild-type and pnrc2 mutant embryos. These data revealed thousands of transcripts are upregulated upon loss of function of pnrc2, among which are transcripts that encode proteins involved in multiple developmental programs and signaling pathways. These results suggest that rapid mRNA decay, facilitated by Pnrc2, may be a mechanism utilized in other developmental contexts to ensure precise spatiotemporal gene regulation during embryogenesis. Lastly, our previously published inducible reporter assays demonstrate that disruption of the AU-rich element (ARE) and Pumilio response element (PRE) in the her1 3’UTR dramatically stabilizes reporter transcripts. To determine the role of the ARE and PRE on endogenous her1 mRNA expression, I explore CRISPR/Cas-mediated knockdown and knockout approaches to downregulate expression of candidate RBPs and disrupt the endogenous motifs in the her1 3’UTR (Chapter 3).
Sharon Amacher (Advisor)
Guramrit Singh (Committee Member)
Aaron Goldman (Committee Member)
Susan Cole (Committee Member)
164 p.
Cellular Biology; Developmental Biology; Molecular Biology
somitogenesis RNA zebrafish development post-transcriptional regulation

Recommended Citations

Citations

  • Blatnik, M. C. (2023). Elucidating Post-transcriptional Regulation of the Vertebrate Segmentation Clock and the Role of Pnrc2-dependent mRNA Decay During Zebrafish Embryogenesis [Doctoral dissertation, Ohio State University]. OhioLINK Electronic Theses and Dissertations Center. http://rave.ohiolink.edu/etdc/view?acc_num=osu1681929063282294

    APA Style (7th edition)

  • Blatnik, Monica. Elucidating Post-transcriptional Regulation of the Vertebrate Segmentation Clock and the Role of Pnrc2-dependent mRNA Decay During Zebrafish Embryogenesis. 2023. Ohio State University, Doctoral dissertation. OhioLINK Electronic Theses and Dissertations Center, http://rave.ohiolink.edu/etdc/view?acc_num=osu1681929063282294.

    MLA Style (8th edition)

  • Blatnik, Monica. "Elucidating Post-transcriptional Regulation of the Vertebrate Segmentation Clock and the Role of Pnrc2-dependent mRNA Decay During Zebrafish Embryogenesis." Doctoral dissertation, Ohio State University, 2023. http://rave.ohiolink.edu/etdc/view?acc_num=osu1681929063282294

    Chicago Manual of Style (17th edition)

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This open access ETD is published by The Ohio State University and OhioLINK.