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"ALTERNATIVE POLYADENYLATION OF PABPN1 AND CSTF-77: INSIGHT INTO TISSUE SPECIFICITY, DIFFERENTIATION, AND MIRNA REGULATION"

by
Lisa K. Hague Canto
Biochemistry and Molecular Biology Program

B.S. 2002, Seton Hall University, South Orange, NJ




Thesis Advisor: Carol S. Lutz, Ph.D.
Associate Professor
Department of Biochemistry and Molecular Biology

Thursday, July 15, 2010
MSB E-609b, 12:00 P.M.


Abstract

Polyadenylation is a 3 end RNA processing event that contributes to the regulation of gene expression by affecting turnover, stability, export, and translation of the message into protein. Approximately fifty-three percent of all human genes have been shown to have more than one polyadenylation site. Regulation at the level of alternative polyadenylation has begun to be investigated, but still requires much more elucidation. Bioinformatic studies have identified unique alternative polyadenylation of two polyadenylation factors, cleavage stimulation factor 77 (CstF-77) and poly (A) binding protein nuclear 1 (PABPN1).
PABPN1 mRNA contains two polyadenylation signals that are utilized in human and mouse cells. The proximal signal is predicted from bioinformatic data to be the strong poly(A) site. The distal polyadenylation signal was suggested to be a weaker site. Additionally, the PABPN1 distal mRNA transcript levels are regulated in a tissue specific manner and in differentiating cells. We present evidence that the 3` untranslated region (3`UTR) of PABPN1 mRNA contains a miRNA-433 binding site that overlaps the core upstream element and leads to reduction of endogenous levels of PABPN1.
The PABPN1 distal poly(A) signal also contains several novel auxiliary upstream sequences. There are two G-rich elements found in the upstream region of PABP mRNA that appear to be very highly conserved, suggestive of important function. Previous studies from other labs have shown that G-rich elements located downstream of polyadenylation sites enhance cleavage and affect polyadenylation signal usage.
The CstF-77 mRNA contains three poly(A) signals, one of which is located in an intron of the CstF-77 gene proximal to the other two signals, which are both located in the 3 most exon. Polyadenylation to yield the shorter transcript would result in a truncated CstF-77 mRNA species. This proximal intronic signal was also shown to be highly conserved in vertebrates, and EST data confirm that this transcript is expressed in both human and mouse cells. Therefore, we are interested in this truncated mRNA, its role in CstF-77 production, and if the intronic transcript is translation competent.


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