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 naturally occurring nucleotides. Given that pseudouridine favors a C-3'-endo structure, our results suggest that it is backbone flexibility that is key to U2AF binding. Indeed, locking the key uridine in the C-3'-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.
Chun Chen,1 Xinliang Zhao,1 Ryszard Kierzek,2 and Yi-Tao Yu1*
Department of Biochemistry and Biophysics, University of Rochester Medical Center, Rochester, New York 14642,1 RNA Chemistry Laboratory, Institute of Bioorganic Chemistry, Polish Academy of Sciences, Noskowskiego 12, Poznan 61-704, Poland2
Molecular and Cellular Biology, September 2010, p. 4108-4119, Vol. 30, No. 17
0270-7306/10/$12.00+0 doi:10.1128/MCB.00531-10
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