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"ENHANCING POLYADENYLATION IN VIVO WITH BIOINFORMATICALLY IDENTIFIED AUXILIARY CIS-ELEMENTS AND PHYLOGENETIC COMPARISON OF HUMAN POLYADENYLATION FACTORS"

by
Sarah Darmon
Biochemistry and Molecular Biology Program
B.S. 2005, Ursinus College



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

Monday, May 21, 2012
10:30 A.M., MSB E-609


Abstract

Polyadenylation is an mRNA processing event that forms the 3’ end of the mRNA. This process contributes to gene expression by affecting stability, export and translation of mRNA. Human polyadenylation signals have both core and auxiliary sequence elements that bind polyadenylation protein factors upstream and downstream of the cleavage site. The majority of mRNAs do not have optimal upstream and downstream core elements, therefore auxiliary elements can aid in polyadenylation efficiency.
While core elements have been studied globally, auxiliary elements were previously identified and studied in a small number of mRNAs. Auxiliary element consensus sequence motifs were identified by a bioinformatic survey in collaboration with our lab (Tian et al., 2005). We used this predicted information to direct our in vivo validation studies. Novel auxiliary elements were placed in a test polyadenylation signal. An in vivo polyadenylation assay determined the strength of the polyadenylation signals when auxiliary elements were included, and compared to a polyadenylation signal without auxiliary elements. In HeLa cells, two of the five novel downstream auxiliary elements enhanced polyadenylation. This assay could not be used for the novel upstream elements but polyadenylation could be measured by the changes in gene expression measured by a luciferase assay. The 3’UTRs containing novel auxiliary elements were placed downstream of the luciferase open reading frame. The same two novel downstream auxiliary elements and all of the novel upstream auxiliary elements had increased reporter protein levels. Therefore, insertion of most of the novel auxiliary elements increased polyadenylation efficiency.
More than half of human genes have more than one polyadenylation signal and undergo alternative polyadenylation (Tian et al., 2005). In an in vivo tandem polyadenylation assay, polyadenylation usage was measured by a vector with a test and a control polyadenylation signal. The addition of all but one novel auxiliary elements increased the polyadenylation usage of the test polyadenylation signal. Therefore, the novel auxiliary elements influence polyadenylation choice.
Well-known auxiliary sequence elements bind specific trans-acting factors that aid in polyadenylation efficiency (Alkan et al., 2006, Arhin et al., 2002, Bagga et al., 1995, Danckwardt et al., 2008, Hall-Pogar et al., 2007, Veraldi et al., 2001). Upstream sequence elements (USEs) are U-rich sequences similar to most of the novel auxiliary upstream elements. Many USEs bind to a variety of proteins including p54 and PSF (Danckwardt et al., 2008, Hall-Pogar et al., 2007). Two novel upstream auxiliary elements bound both trans-acting factors, p54 and PSF, in a biotinylated pull-down assay. siRNA knockdown of p54 and PSF eliminated the enhancement of the novel upstream auxiliary elements to at least wild type levels in a luciferase assay. Therefore, novel auxiliary upstream elements bind trans-acting factors such as p54 and PSF that aid in polyadenylation.
Since polyadenylation plays an important role in the biogenesis of mammalian mRNAs, we wanted to investigate how conserved the factors responsible for carrying out this process in other species. We found that homologs of human polyadenylation factors were maintained in protein length, sequence and domains throughout the majority of the vertebrates and insects. These species are predicted to have a similar mechanisms of polyadenylation to that found in humans. The differences in factors and domains occurred most often in plants, trypanosomes and yeast. Therefore, these species are likely to have subtle different mechanisms of polyadenylation compared to human. Overall, the polyadenylation factors are conserved evolutionary, suggesting that the mechanism is ancient.


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