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A Molecular, Evolutionary and Functional Study of RNP-4F Splicing Assembly Factor Gene Expression in Drosophila melanogaster

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2016, Doctor of Philosophy, Miami University, Cell, Molecular and Structural Biology (CMSB).
In the first chapter I have discussed the advantages of Drosophila as a model organism, the ΦC31 transgenesis system, various aspects of splicing and splice factors and role of RNA in regulating gene expression. Chapter 2 is a detailed study of the truncated dADAR protein during Drosophila embryogenesis. 3’-RACE has shown that during D. melanogaster embryogenesis truncated dADAR mRNA levels are developmentally controlled. Western analysis showed that both full-length and truncated dADAR isoform classes are translated into protein in all Drosophila species used in the study. RNAi to specifically knock down the full-length dADAR transcript caused no changes to the long rnp-4f mRNA levels, suggesting an evolutionarily-conserved function of the truncated dADAR in regulating alternative splicing of rnp-4f during Drosophila embryogenesis. Chapter 3 includes a description of the ΦC31 based rnp-4f – GFP reporter gene expression vectors. Using the UAS-Gal4 system, we showed that presence of the regulatory rnp-4f stem-loop enhances reporter expression, which is further enhanced by overexpression of the RNP-4F transgene. We have proposed a model which suggests that RNP-4F protein acts as an enhancement factor and binds to its own mRNA 5’-UTR to enhance protein synthesis in the developing fly CNS by positive autoregulation. Chapter 4 is a study of an affinity-based approach to isolate RNA probe UV-crosslinked to embryo protein, to investigate the composition of the dsRNA binding protein complex which regulates rnp-4f gene expression. The RNA-protein complex was analyzed by MALDI-TOF MS and several protein candidates were identified. In Chapter 5, I have discussed the research objectives accomplished in my dissertation. I have mentioned future goals designed to further our understanding of rnp-4f and dADAR regulation during Drosophila embryogenesis. The last section includes the appendices divided into three parts. The first part is a REMSA based study to show that dADAR binds to the long rnp-4f 5’-UTR stem-loop to regulate alternative splicing. The second appendix is a study of the RNP-4F associated U4-U6 snRNA secondary structure and the structural features of the rnp-4f mRNA. The final chapter is a detailed report of the reagent composition and protocols used in all the experiments.
Jack Vaughn (Advisor)
Susan Hoffman (Committee Member)
David Pennock (Committee Member)
Haifei Shi (Committee Member)
Xiao-Wen Cheng (Committee Member)

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Citations

  • Ghosh, S. (2016). A Molecular, Evolutionary and Functional Study of RNP-4F Splicing Assembly Factor Gene Expression in Drosophila melanogaster [Doctoral dissertation, Miami University]. OhioLINK Electronic Theses and Dissertations Center. http://rave.ohiolink.edu/etdc/view?acc_num=miami1465473655

    APA Style (7th edition)

  • Ghosh, Sushmita. A Molecular, Evolutionary and Functional Study of RNP-4F Splicing Assembly Factor Gene Expression in Drosophila melanogaster . 2016. Miami University, Doctoral dissertation. OhioLINK Electronic Theses and Dissertations Center, http://rave.ohiolink.edu/etdc/view?acc_num=miami1465473655.

    MLA Style (8th edition)

  • Ghosh, Sushmita. "A Molecular, Evolutionary and Functional Study of RNP-4F Splicing Assembly Factor Gene Expression in Drosophila melanogaster ." Doctoral dissertation, Miami University, 2016. http://rave.ohiolink.edu/etdc/view?acc_num=miami1465473655

    Chicago Manual of Style (17th edition)