Department of Biochemistry and Biophysics, Center for RNA Biology, University of Rochester School of Medicine and Dentistry, Rochester, NY, 14642, USA.
Department of Biochemistry and Biophysics, Center for RNA Biology, University of Rochester School of Medicine and Dentistry, Rochester, NY, 14642, USA.
Biochem Biophys Res Commun. 2019 Apr 2;511(2):416-421. doi: 10.1016/j.bbrc.2019.02.073. Epub 2019 Feb 21.
The pre-mRNA branch point sequence (BPS) anneals with a pseudouridine-modified region of the U2 small nuclear (sn)RNA, and offers a 2' hydroxyl group of a bulged adenosine as the nucleophile for the first catalytic step of pre-mRNA splicing. To increase our structural understanding of branch site selection, we characterized a duplex containing a BPS sequence that is common among multicellular eukaryotes (5'-UACUGAC-3') and the complementary U2 snRNA site using NMR. A major conformation of the expected branch site adenosine stacked within the duplex and paired with the conserved pseudouridine of the U2 snRNA strand. In contrast, the guanosine preceding the branch site appeared flexible and had weak contacts with the surrounding nucleotides. Pseudouridine-modified and unmodified U2 snRNA-BPS-containing duplexes remained structurally similar. These results highlight the importance of auxiliary factors to achieve the active bulged conformation of the branch site nucleophile for the first step of pre-mRNA splicing.
前体 mRNA 分支点序列 (BPS) 与 U2 小核 (sn)RNA 的假尿嘧啶修饰区域退火，并提供凸起的腺苷的 2' 羟基作为前体 mRNA 剪接的第一步的亲核试剂。为了提高我们对分支位点选择的结构理解，我们使用 NMR 对包含常见于多细胞真核生物的 BPS 序列 (5'-UACUGAC-3') 和互补的 U2 snRNA 位点的双链体进行了表征。预期的分支位点腺苷的主要构象在双链体内堆积，并与 U2 snRNA 链的保守假尿嘧啶配对。相比之下，分支位点前的鸟嘌呤显得灵活，与周围核苷酸的接触较弱。经过假尿嘧啶修饰和未经修饰的 U2 snRNA-BPS 双链体保持结构相似。这些结果突出了辅助因子对于实现分支位点亲核试剂的活性凸起构象以进行前体 mRNA 剪接的第一步的重要性。
