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dc.creatorShankarling, Ganesh S.
dc.date.available2011-02-18T22:12:51Z
dc.date.issued2009-05
dc.identifier.urihttp://hdl.handle.net/2346/17778en_US
dc.description.abstractAlternative splicing and polyadenylation are important mechanisms for creating the proteomic diversity necessary for the nervous system to fulfill its specialized functions. I discovered an evolutionarily conserved family of alternatively spliced mRNAs encoding the CstF-64 polyadenylation protein collectively called βCstF-64 that could potentially contribute to proteomic diversity in the nervous system. The βCstF-64 variant mRNA in mice was generated by inclusion of two alternate exons (that we call exons 8.1 and 8.2) found between exons 8 and 9 of the CstF-64 gene, and contained an additional 147 nucleotides, encoding 49 additional amino acids. Immunoblot and 2D-PAGE analyses of mouse nuclear extracts showed that a protein corresponding to βCstF-64 was expressed in brain at approximately equal levels to CstF-64. I also found that βCstF-64 was expressed in all parts of the brain, the spinal cord, and in neuron-like cell lines including PC-12 cells, where its expression was regulated by nerve growth factor. These data together with the extensive conservation of the βCstF-64 splice variant family members in vertebrate species suggested an evolutionarily conserved function for βCstF-64 in neural gene expression. I hypothesized that βCstF-64 functioned in polyadenylation of nervous system-expressed mRNAs. In order to test the hypothesis that βCstF-64 played a role in mRNA polyadenylation in the nervous system, I chose to focus on establishing that βCstF-64 was a polyadenylation factor. Co-immunoprecipitation analysis indicated that βCstF-64 was part of the CstF complex and hence a polyadenylation protein by this criteria. I used in vivo luciferase assay to test whether βCstF-64 could promote polyadenylation of reporter genes. For this, I used the β-adducin mRNA as a model since it contained two prominent evolutionarily conserved poly(A) sites (termed pA1 and pA4), with the promoter-distal pA4 site being brain-specific. With this assay, I showed that βCstF-64 was as active as CstF-64 in enhancing luciferase activity from plasmids containing polyadenylation signals corresponding to the pA1 and pA4 sites of β-adducin mRNA. These data supported the hypothesis that βCstF-64, like CstF-64, was a polyadenylation protein. In contrast to the observations in PC-12 cells, βCstF-64 was less active than CstF-64 in enhancing luciferase activity from plasmids containing the brain-specific pA4 polyadenylation region of β-adducin mRNA in HeLa cells (a non-neuronal cell line). These data led me to propose that βCstF-64 interacts with neuronal proteins that modulate its activity on certain polyadenylation sites in neuronal cells. Our discovery and functional characterization of βCstF-64 is of importance to the field of RNA processing since it is the first instance of a nervous system-specific isoform of a key polyadenylation protein. This discovery has paved way for future studies to help understand the role of βCstF-64 in neural mRNA polyadenylation and to uncover potential new mechanisms of alternative RNA processing in the nervous system.
dc.format.mimetypeapplication/pdf
dc.language.isoeng
dc.publisherTexas Tech Universityen_US
dc.subjectCleavage stimulation factor (CstF-64)en_US
dc.subjectSplice varianten_US
dc.subjectPolyadenylationen_US
dc.titleFunctional characterization of a nervous system-expressed isoform of the CstF-64 polyadenylation protein
dc.typeDissertation
thesis.degree.namePh.D.
thesis.degree.levelDoctoral
thesis.degree.disciplineTTUHSC -- Biochemistry and Molecular Genetics
thesis.degree.grantorTexas Tech University
thesis.degree.departmentTTUHSC -- Biochemistry and Molecular Genetics
dc.contributor.committeeMemberSchneider, Brandt
dc.contributor.committeeMemberWilliams, Simon C.
dc.contributor.committeeMemberWhelly, Sandra M.
dc.contributor.committeeMemberCoates, Penelope
dc.contributor.committeeChairMacDonald, Clinton C.
dc.degree.departmentTTUHSC -- Biochemistry and Molecular Geneticsen_US
dc.rights.availabilityUnrestricted.


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