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Understanding the Role of Exon Junction Complex-dependent Nonsense Mediated mRNA Decay in Zebrafish Embryonic Development

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2019, Doctor of Philosophy, Ohio State University, Molecular Genetics.

Post-transcriptional control of gene expression is essential for proper development and is achieved largely by RNA-binding proteins. My graduate research has focused on understanding the role of one such RNA binding protein complex, the Exon Junction Complex (EJC) in development. EJC is deposited about 24 nts upstream of exon-exon junctions during splicing. EJC influences many aspects of post-transcriptional regulation such as mRNA splicing, export, localization and nonsense mediated mRNA decay (NMD). EJC-dependent NMD is recognized as one mode of rapidly regulating gene expression of normal mRNAs that contain NMD-inducing features such as 3′UTR intron or an upstream ORF. How regulation of gene expression via EJC-dependent NMD influences development of specific tissues is unknown. My work utilizes a strong developmental and genetic model system, zebrafish, to understand how EJC-dependent NMD shapes development.

In order to address my scientific questions, I utilized the RNA-Seq technique. Our lab has a custom RNA-Seq library preparation protocol which I wanted to improve in order to increase the efficiency of isolating sample cDNA after reverse transcription of RNA fragments (Chapter 2). To improve the protocol, I incorporated biotinylated dNTPs in the RT reaction so that cDNA generated from RNA fragments could be extracted using streptavidin beads. This amendment to the library preparation protocol efficiently selects for extended RT product and avoids ligation of the unextended adapter and generation of insert-lacking cDNAs in the library.

To study EJC function during development (Chapter 3), I generated zebrafish mutants in EJC core protein genes rbm8a and magoh. Homozygous rbm8a and magoh mutants (EJC mutants) are paralyzed and have muscle and neural defects. As expected, RNA profiling revealed that annotated aberrant and normal NMD targets are significantly upregulated in EJC mutants. An mRNA is targeted for NMD by the key NMD-regulator Upf1 when an exon-exon junction, marked by the EJC, is bound ≥ 50 nts downstream of the terminated ribosome. Surprisingly, I discovered that some upregulated normal transcripts contain a conserved proximal 3′ UTR intron (3′UI) < 50 nts downstream of the stop codon. These ‘proximal 3′UI-containing NMD targets’ are similarly up-regulated in Upf1-deficient and NMD inhibitor-treated embryos, suggesting that this subset of rbm8a- and magoh-regulated transcripts is regulated via NMD. The same trend is observed in Upf1-deficient mammalian cells. One proximal 3′UI+ NMD target which is upregulated in EJC mutants and upf1 morphants encodes Foxo3b. I find that heterozygous and homozygous knockout of foxo3b partially rescues motor neuron outgrowth defects in EJC mutants. My work establishes zebrafish as a system to study NMD, characterizes zebrafish embryonic muscle and neural defects associated with loss of EJC and identifies proximal 3′UTR intron-containing genes a new class of NMD targets.

Sharon Amacher (Advisor)
Guramrit Singh (Advisor)
Robin Wharton (Committee Member)
Anita Hopper (Committee Member)
Beattie Christine (Committee Member)
159 p.

Recommended Citations

Citations

  • Gangras, P. (2019). Understanding the Role of Exon Junction Complex-dependent Nonsense Mediated mRNA Decay in Zebrafish Embryonic Development [Doctoral dissertation, Ohio State University]. OhioLINK Electronic Theses and Dissertations Center. http://rave.ohiolink.edu/etdc/view?acc_num=osu1574765848602854

    APA Style (7th edition)

  • Gangras, Pooja. Understanding the Role of Exon Junction Complex-dependent Nonsense Mediated mRNA Decay in Zebrafish Embryonic Development. 2019. Ohio State University, Doctoral dissertation. OhioLINK Electronic Theses and Dissertations Center, http://rave.ohiolink.edu/etdc/view?acc_num=osu1574765848602854.

    MLA Style (8th edition)

  • Gangras, Pooja. "Understanding the Role of Exon Junction Complex-dependent Nonsense Mediated mRNA Decay in Zebrafish Embryonic Development." Doctoral dissertation, Ohio State University, 2019. http://rave.ohiolink.edu/etdc/view?acc_num=osu1574765848602854

    Chicago Manual of Style (17th edition)