Probing interactions between the U2 small nuclear ribonucleoprotein and the DEAD-box protein, Prp5.
Probing interactions between the U2 small nuclear ribonucleoprotein and the DEAD-box protein, Prp5.
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DOI：
10.1074/jbc.m109553200
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发表时间：
2002-06-07
影响因子：
4.8
通讯作者：
Ruby, SW
中科院分区：
文献类型：
--
作者：
Abu Dayyeh, BK;Quan, TK;Ruby, SW
前mRNA在前剪接体形成过程中与酵母U2小核核糖核蛋白（snRNP）结合需要ATP水解、前mRNA的分支点区域的高度保守的UACUAAC盒以及若干因子。在这里，我们分析了与U2小核RNA互补的放射性标记的2 '-O-甲基寡核苷酸的结合，以研究UACUAAC盒、U2 snRNP和Prp 5 p（一种前剪接体形成所必需的DEAD盒蛋白）之间的相互作用。通过凝胶电泳测定2 '-O-甲基寡核苷酸与酵母细胞提取物中U2 snRNP的结合。结合是快速的，增强ATP，并依赖于U2 snRNP的完整性和构象。它也被Prp 5 p刺激，Prp 5 p被发现与U2 snRNP物理相关。在体外热灭活的温度敏感的prp 5 -1突变体提取物减少寡核苷酸结合U2和ATP增强结合3倍。此外，温度敏感的prp 5 -1突变映射到解旋酶样结构域内的ATP结合基序I。因此，Prp 5 p的催化活性可能促进U2 snRNP的构象变化。
Pre-mRNA binding to the yeast U2 small nuclear ribonucleoprotein (snRNP) during prespliceosome formation requires ATP hydrolysis, the highly conserved UACUAAC box of the branch point region of the pre-mRNA, and several factors. Here we analyzed the binding of a radiolabeled 2'-O-methyl oligonucleotide complementary to U2 small nuclear RNA to study interactions between the UACUAAC box, U2 snRNP, and Prp5p, a DEAD box protein necessary for prespliceosome formation. Binding of the 2'-O-methyl oligonucleotide to the U2 snRNP in yeast cell extract was assayed by gel electrophoresis. Binding was rapid, enhanced by ATP, and dependent on the integrity and conformation of the U2 snRNP. It was also stimulated by Prp5p that was found to associate physically with U2 snRNP. In vitro heat inactivation of the temperature-sensitive prp5-1 mutant extract decreased oligonucleotide binding to U2 and the ATP enhancement of binding by 3-fold. Furthermore, the temperature-sensitive prp5-1 mutation maps to the ATP-binding motif I within the helicase-like domain. Thus the catalytic activity of Prp5p likely promotes a conformational change in the U2 snRNP.