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"REGULATION OF ALTERNATIVE POLYADENYLATION BY
TRANSCRIPTION, POLYADENYLATION, AND SPLICING PARAMETERS"

by
Wenting Luo
Biochemistry and Molecular Biology Program
B.S. 2003, Jinan University



Thesis Advisor: Bin Tian, Ph.D.
Associate Professor
Department of Biochemistry and Molecular Biology

Thursday, April 26, 2012
2:00 P.M., MSB E-609


Abstract

Alternative polyadenylation leads to mRNA isoforms containing different coding sequences (CDS) and/or 3f untranslated regions (3fUTRs), and has been shown to be dynamically regulated in different biological settings.

A protein-coding gene can have multiple polyA sites located in different regions. The 3f-most polyA site defines the 3f end of the gene, and typically results in the longest mRNA isoform. Other polyA sites located in the same exon lead to isoforms with shorter 3fUTRs. In addition, some polyA sites are located in regions that can be removed from the nascent RNA by splicing, which are termed intronic polyA sites. Intronic polyA sites can also lead to mRNA isoforms with different CDS. Moreover, polyA sites can be located within the CDS, giving rise to transcripts without an in-frame stop codon.

By systematic analysis of human and mouse transcriptomes, we found that short 3UTR isoforms are relatively more abundant when genes are highly expressed whereas long 3UTR isoforms are relatively more abundant when genes are lowly expressed. Reporter assays indicated that polyA site choice can be modulated by transcriptional activity through the gene promoter. Using global and reporter-based nuclear run-on assays, we found that RNA polymerase II is more likely to pause at the polyA site of highly expressed genes than that of lowly expressed ones. Moreover, highly expressed genes tend to have a lower level of nucleosome but higher H3K4me3 and H3K36me3 levels at promoter-proximal polyA sites relative to distal ones. Taken together, our results indicate that polyA site usage is generally coupled to transcriptional activity, leading to regulation of alternative polyadenylation by transcription.

The human gene encoding the polyadenylation factor CstF-77 contains 21 exons. However, intron 3 accounts for over half of the gene region. In contrast to the trend that large introns in the human genome have strong 5f splice site (5fSS), this intron has a weak 5fSS. In addition, a polyA site with medium strength is located in the intron, leading to truncated mRNA products. Significantly, the gene structure, intron size, and strengths of 5fSS, 3fSS, and polyA site are all highly conserved across vertebrates. Perturbation of these parameters can dramatically alter the kinetics of usage of the intronic polyA site. We found that the truncated mRNAs resulting from intronic polyadenylation in human cells does not lead to detectable protein products, most likely due to rapid protein degradation, and the usage of the intronic polyA site is responsive to the CstF-77 expression level, suggesting that intronic polyadenylation is employed to attenuate the expression of CstF-77 via a negative feed-back auto-regulatory mechanism. By analyzing a large set of gene expression data from a diverse array of biological conditions and validating in the C2C12 myoblast differentiation, we found that the usage of intronic polyA site is sensitive to the general splicing activity of the cell, suggesting a mechanism by which polyadenylation activity coordinates with splicing activity. Thus, the conserved intronic polyA site in the CstF-77 gene is a sensor which modulates cellular polyadenylation activity and balances polyadenylation with splicing.


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