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TitleStudies of polyadenylation regulation of U1A mRNA by an RNP complex containing U1A and U1 snRNP
NameCaratozzolo, Rose Marie (author), GUNDERSON, SAMUEL I (chair), Brewer, Gary (internal member), Covey, Lori (internal member), Tian, Bin (outside member), Rutgers University, Graduate School - New Brunswick,
Degree Date2011-01
Date Created2011
SubjectBiochemistry,
Gene expression,
Genetic regulation,
RNA
DescriptionThe 3’-end processing of nearly all eukaryotic pre-mRNAs comprises multiple steps which culminate in the addition of a poly(A) tail, which is essential for mRNA stability, translation, and export. Consequently, polyadenylation regulation is an important component of gene expression. One way to regulate polyadenylation is to inhibit the activity of a single poly(A) site, as exemplified by the U1A protein that negatively autoregulates itself by binding to a Polyadenylation Inhibitory Element (PIE) site within the 3’ UTR of its own pre-mRNA. U1 snRNP, which is primarily involved in splice site recognition, inhibits poly(A) site activity in papillomaviruses by binding to 5’ splice site-like sequences, which have recently been named “U1-sites”. Here, a recently identified U1-site in the human U1A 3'UTR is examined and shown to synergize with the adjacent PIE site to inhibit polyadenylation. However, unlike the sites found in papillomaviruses, the U1A U1-site has no inhibitory activity on its own and is dependent on a wild-type PIE. This lack of activity is due to the site being masked within a phylogenetically conserved stem structure (U1-STEM). The secondary RNA structure of this region was confirmed by RNase digestion analysis. Mutation of the U1-STEM, thereby opening up the U1-site, greatly increases U1-site mediated inhibition. The region between the U1-STEM and PIE (referred to as Region C) was also revealed to be required for synergy. Since biotin pulldown assays indicated that U1 snRNP binding to the U1-site was not affected by the presence of the U1-STEM, a model was proposed suggesting that U1 snRNP binds to the U1-STEM, but remains trapped in an inactive conformation until PIE is bound by two U1A molecules. However, further experiments showed that U1 snRNP binding did actually increase when the U1-STEM was mutated, but no corresponding change to the U1-STEM structure was detected. The discrepancies within these data suggest there is still much to be determined regarding the binding of U1 snRNP to the U1-Site. A more refined model is then presented which involves remodeling of Region C and part of the U1-STEM.
NotePh.D.
NoteIncludes bibliographical references
NoteIncludes vita
Noteby Rose Marie Caratozzolo
Genretheses
Persistent URLhttp://hdl.rutgers.edu/1782.1/rucore10001600001.ETD.000057530
Languageeng
CollectionGraduate School - New Brunswick Electronic Theses and Dissertations
Organization NameRutgers, The State University of New Jersey
RightsThe author owns the copyright to this work.
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