Mechanisms and Functional Roles of Pseudouridines in RNAs

Thesis (Ph.D.)--University of Rochester. School of Medicine & Dentistry. Dept. of Biochemistry & Biophysics, 2010.Pre-mRNA splicing is an essential RNA processing reaction by which intervening sequences (introns) are removed and protein-coding sequences (exons) are ligated together. Pre-mRNA splicing is carried out by the spliceosome, a large RNA-protein complex consisting of 5 small uridine-rich RNAs (U1, U2, U4, U5 and U6 snRNA) and many proteins. Several consensus sequences in intron are required for the precise removal of introns during pre-mRNA splicing, the 5’ splice site (5’SS), the branch point and the 3’ splice site (3’SS) with an adjacent polypyrimidine tract in higher eukaryotes. The polypyrimidine tract near the 3’ splice site is important for pre-mRNA splicing. Using pseudouridine incorporation and in vivo RNA-guided RNA pseudouridylation, we have identified two important uridines in the polypyrimidine tract of adenovirus pre-mRNA. Conversion of either uridine into pseudouridine leads to a splicing defect in Xenopus oocytes. Using a variety of molecular biology methodologies, we show that the splicing defect is due to the failure of U2AF65 to recognize the pseudouridylated polypyrimidine tract. This negative impact on splicing is pseudouridine-specific, as no effect is observed when the uridine is changed to other natural occurring nucleotides. Given that pseudouridine favors a C3’-endo structure, our results suggest that it is backbone flexibility that is key to U2AF binding. Indeed, locking the key uridine in the C3’-endo configuration while maintaining its uridine identity blocks U2AF65 binding and splicing. This pseudouridine effect can also be applied to other pre-mRNA polypyrimidine tracts. Thus, our work demonstrates that in vivo binding of U2AF65 to a polypyrimidine tract requires a flexible RNA backbone. U1 snRNA is involved in recognition of the 5'SS through complementary base-pairing interactions. Interestingly, the 5' region of U1 snRNA responsible for recognition of the 5'SS contains two post-transcriptionally modified uridines (5 and 6). Furthermore, these two modifications are evolutionarily conserved from yeast to humans. Preliminary studies in S. cerevisiae indicate that Cbf5p, a pseudouridine synthase, and Nhp2p, one of the core proteins from box H/ACA RNPs, are involved in the formation of 5 and 6 in yeast U1 snRNA. An in vitro pseudouridylation assay also suggests that 6 formation is RNA-dependent, further supporting the notion that this particular modification is introduced by a box H/ACA RNP. However, the set of predicted guide RNAs responsible for the two modifications are experimentally determined to be incorrect