A flexible RNA backbone within the polypyrimidine tract is required for U2AF65 binding and pre-mRNA splicing in vivo.
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 U2AF(65) 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 U2AF(65) 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 U2AF(65) to a polypyrimidine tract requires a flexible RNA backbone.
Chen
Chun
C
Department of Biochemistry and Biophysics, University of Rochester Medical Center, Rochester, NY 14642, USA.
Zhao
Xinliang
X
Kierzek
Ryszard
R
Yu
Yi-Tao
YT
D000818:Animals; D001483:Base Sequence; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000378:metabolism*; D009690:Nucleic Acid Conformation; D009865:Oocytes / Q000378:metabolism; D011485:Protein Binding; D011743:Pyrimidines / Q000737:chemistry / Q000378:metabolism*; D012322:RNA Precursors / Q000737:chemistry / Q000378:metabolism*; D012326:RNA Splicing; D012261:Ribonucleoproteins / Q000378:metabolism*; D000072668:Splicing Factor U2AF; D014529:Uridine / Q000737:chemistry / Q000378:metabolism; D014981:Xenopus
D009687:Nuclear Proteins; D011743:Pyrimidines; D012322:RNA Precursors; D012261:Ribonucleoproteins; D000072668:Splicing Factor U2AF; C030986:pyrimidine; D014529:Uridine