Functional characterization of a nervous system-expressed isoform of the CstF-64 polyadenylation protein
Shankarling, Ganesh S.
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Alternative 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 Î²CstF-64 that could potentially contribute to proteomic diversity in the nervous system. The Î²CstF-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 Î²CstF-64 was expressed in brain at approximately equal levels to CstF-64. I also found that Î²CstF-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 Î²CstF-64 splice variant family members in vertebrate species suggested an evolutionarily conserved function for Î²CstF-64 in neural gene expression. I hypothesized that Î²CstF-64 functioned in polyadenylation of nervous system-expressed mRNAs. In order to test the hypothesis that Î²CstF-64 played a role in mRNA polyadenylation in the nervous system, I chose to focus on establishing that Î²CstF-64 was a polyadenylation factor. Co-immunoprecipitation analysis indicated that Î²CstF-64 was part of the CstF complex and hence a polyadenylation protein by this criteria. I used in vivo luciferase assay to test whether Î²CstF-64 could promote polyadenylation of reporter genes. For this, I used the Î²-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 Î²CstF-64 was as active as CstF-64 in enhancing luciferase activity from plasmids containing polyadenylation signals corresponding to the pA1 and pA4 sites of Î²-adducin mRNA. These data supported the hypothesis that Î²CstF-64, like CstF-64, was a polyadenylation protein. In contrast to the observations in PC-12 cells, Î²CstF-64 was less active than CstF-64 in enhancing luciferase activity from plasmids containing the brain-specific pA4 polyadenylation region of Î²-adducin mRNA in HeLa cells (a non-neuronal cell line). These data led me to propose that Î²CstF-64 interacts with neuronal proteins that modulate its activity on certain polyadenylation sites in neuronal cells. Our discovery and functional characterization of Î²CstF-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 Î²CstF-64 in neural mRNA polyadenylation and to uncover potential new mechanisms of alternative RNA processing in the nervous system.