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Singh Jagjit Ph.D. Thesis .pdf (4.23 MB)

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RNA-Protein Interactions in the U12-Dependent Spliceosome

Singh, Jagjit
http://rave.ohiolink.edu/etdc/view?acc_num=csu1484307043050366

Abstract Details

2016, Doctor of Philosophy in Regulatory Biology, Cleveland State University, College of Sciences and Health Professions.
Nuclear precursor messenger RNA (Pre-mRNA) splicing is an important regulatory step in metazoan gene expression. More than 99% of nuclear pre-mRNA introns are U2-type that are spliced by U2-dependent spliceosome containing U1, U2, U4, U5 and U6 snRNAs. Only less than 1% of the introns are U12-type and spliced by U11, U12, U4atac, U5 and U6atac snRNAs. U12 and U6atac snRNAs play a central role in the splicing of U12-dependent introns. Our previous work demonstrated that the conserved 3' stem-loop region of U6atac snRNA contains a U12-dependent spliceosome-specific targeting activity, however any potential molecular mechanism was unclear. We discovered that the distal 3' stem-loop of U6atac has structural and sequence similarities with stem-loop III of U12 snRNA. These observations convinced us to investigate the structure-function requirement of the substructure of the U6atac 3' stem-loop in U12-dependent in vivo splicing. Our results show that the C-terminal RNA recognition motif of p65, a U12 snRNA binding protein, also binds to the distal 3' stem-loop of U6atac. Using in vivo genetic suppressor assay, we demonstrate that stem-loop III of U12 snRNA which binds to p65 protein can be functionally replaced by U6atac distal stem-loop and vice-versa. Furthermore, we tested the compatibility of the U6atac 3' end from phylogenetically distant species in a human U6atac suppressor background to establish the evolutionary relatedness of these structures and in vivo functionality. In conclusion, we demonstrate that p65 C-terminal RNA recognition motif interacts with the U6atac distal 3' stem-loop. Although the significance of p65 binding to U6atac snRNA is not clear, our study suggests that both the helix structure, as well as the sequence of U6atac distal 3' stem-loop is important for snRNA-protein interactions and U12-dependent intron splicing.
Girish Shukla, Ph.D. (Advisor)
Barsanjit Mazumder, Ph.D. (Committee Member)
Sailen Barik, Ph.D. (Committee Member)
Aaron Severson, Ph.D. (Committee Member)
Sadashiva Karnik, Ph.D. (Committee Member)
Anton Komar, Ph.D. (Committee Member)
Richard Padgett, Ph.D. (Committee Member)
145 p.
Molecular Biology
U12 snRNA, U6atac snRNA and p65 protein

Recommended Citations

Citations

  • Singh, J. (2016). RNA-Protein Interactions in the U12-Dependent Spliceosome [Doctoral dissertation, Cleveland State University]. OhioLINK Electronic Theses and Dissertations Center. http://rave.ohiolink.edu/etdc/view?acc_num=csu1484307043050366

    APA Style (7th edition)

  • Singh, Jagjit. RNA-Protein Interactions in the U12-Dependent Spliceosome . 2016. Cleveland State University, Doctoral dissertation. OhioLINK Electronic Theses and Dissertations Center, http://rave.ohiolink.edu/etdc/view?acc_num=csu1484307043050366.

    MLA Style (8th edition)

  • Singh, Jagjit. "RNA-Protein Interactions in the U12-Dependent Spliceosome ." Doctoral dissertation, Cleveland State University, 2016. http://rave.ohiolink.edu/etdc/view?acc_num=csu1484307043050366

    Chicago Manual of Style (17th edition)

csu1484307043050366
453
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This open access ETD is published by Cleveland State University and OhioLINK.