Polyadenylation site-specific differences in the activity of the neuronal ßCstF-64 protein in PC-12 cells.

Recent genome-wide analyses have implicated alternative polyadenylation - the process of regulated mRNA 3' end formation - as a critical mechanism that influences multiple steps of mRNA metabolism in addition to increasing the protein-coding capacity of the genome. Although the functional consequences of alternative polyadenylation are well known, protein factors that regulate this process are poorly characterized. Previously, we described an evolutionarily conserved family of neuronal splice variants of the CstF-64 mRNA, ßCstF-64, that we hypothesized to function in alternative polyadenylation in the nervous system. In the present study, we show that ßCstF-64 mRNA and protein expression increase in response to nerve growth factor (NGF), concomitant with differentiation of adrenal PC-12 cells into a neuronal phenotype, suggesting a role for ßCstF-64 in neuronal gene expression. Using PC-12 cells as model, we show that ßCstF-64 is a bona fide polyadenylation protein, as evidenced by its association with the CstF complex, and by its ability to stimulate polyadenylation of luciferase reporter mRNA. Using luciferase assays, we show that ßCstF-64 stimulates polyadenylation equivalently at the two weak poly(A) sites of the ß-adducin mRNA. Notably, we demonstrate that the activity of ßCstF-64 is less than CstF-64 on a strong polyadenylation signal, suggesting polyadenylation site-specific differences in the activity of the ßCstF-64 protein. Our data address the polyadenylation functions of ßCstF-64 for the first time, and provide initial insights into the mechanism of alternative poly(A) site selection in the nervous system.

The τCstF-64 polyadenylation protein controls genome expression in testis.

The τCstF-64 polyadenylation protein (gene symbol Cstf2t) is a testis-expressed orthologue of CstF-64. Mice in which Cstf2t was knocked out had a phenotype that was only detected in meiotic and postmeiotic male germ cells, giving us the opportunity to examine CstF-64 function in an isolated developmental system. We performed massively parallel clonally amplified sequencing of cDNAs from testes of wild type and Cstf2t(-/-) mice. These results revealed that loss of τCstF-64 resulted in large-scale changes in patterns of genome expression. We determined that there was a significant overrepresentation of RNAs from introns and intergenic regions in testes of Cstf2t(-/-) mice, and a concomitant use of more distal polyadenylation sites. We observed this effect particularly in intronless small genes, many of which are expressed retroposons that likely co-evolved with τCstF-64. Finally, we observed overexpression of long interspersed nuclear element (LINE) sequences in Cstf2t(-/-) testes. These results suggest that τCstF-64 plays a role in 3' end determination and transcription termination for a large range of germ cell-expressed genes.

A family of splice variants of CstF-64 expressed in vertebrate nervous systems.

BACKGROUND: Alternative splicing and polyadenylation are important mechanisms for creating the proteomic diversity necessary for the nervous system to fulfill its specialized functions. The contribution of alternative splicing to proteomic diversity in the nervous system has been well documented, whereas the role of alternative polyadenylation in this process is less well understood. Since the CstF-64 polyadenylation protein is known to be an important regulator of tissue-specific polyadenylation, we examined its expression in brain and other organs. RESULTS: We discovered several closely related splice variants of CstF-64 - collectively called betaCstF-64 - that could potentially contribute to proteomic diversity in the nervous system. The betaCstF-64 splice variants are found predominantly in the brains of several vertebrate species including mice and humans. The major betaCstF-64 variant mRNA is 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 contains an additional 147 nucleotides, encoding 49 additional amino acids. Some variants of betaCstF-64 contain only the first alternate exon (exon 8.1) while other variants contain both alternate exons (8.1 and 8.2). In mice, the predominant form of betaCstF-64 also contains a deletion of 78 nucleotides from exon 9, although that variant is not seen in any other species examined, including rats. Immunoblot and 2D-PAGE analyses of mouse nuclear extracts indicate that a protein corresponding to betaCstF-64 is expressed in brain at approximately equal levels to CstF-64. Since betaCstF-64 splice variant family members were found in the brains of all vertebrate species examined (including turtles and fish), this suggests that betaCstF-64 has an evolutionarily conserved function in these animals. betaCstF-64 was present in both pre- and post-natal mice and in different regions of the nervous system, suggesting an important role for betaCstF-64 in neural gene expression throughout development. Finally, experiments in representative cell lines suggest that betaCstF-64 is expressed in neurons but not glia. CONCLUSION: This is the first report of a family of splice variants encoding a key polyadenylation protein that is expressed in a nervous system-specific manner. We propose that betaCstF-64 contributes to proteomic diversity by regulating alternative polyadenylation of neural mRNAs.