Graduate Program, Department of Chemistry and Biochemistry, University of Texas at Austin, Austin, Texas 78712, USA.
RNA. 2010 Mar;16(3):516-28. doi: 10.1261/rna.2030510. Epub 2010 Jan 20.
Eukaryotic pre-mRNA splicing is a complex process requiring the precise timing and action of >100 trans-acting factors. It has been known for some time that the two steps of splicing chemistry require three DEAH-box RNA helicase-like proteins; however, their mechanism of action at these steps has remained elusive. Spliceosomes arrested in vivo at the three helicase checkpoints were purified, and first step-arrested spliceosomes were functionally characterized. We show that the first step of splicing requires a novel ATP-independent conformational change. Prp2p then catalyzes an ATP-dependent rearrangement displacing the SF3a and SF3b complexes from the branchpoint within the spliceosome. We propose a model in which SF3 prevents premature nucleophilic attack of the chemically reactive hydroxyl of the branchpoint adenosine prior to the first transesterification. When the spliceosome attains the proper conformation and upon the function of Prp2p, SF3 is displaced from the branchpoint allowing first step chemistry to occur.
真核生物前体 mRNA 的剪接是一个复杂的过程，需要 >100 种反式作用因子的精确定时和作用。一段时间以来，人们已经知道剪接化学反应的两个步骤需要三种 DEAH-box RNA 解旋酶样蛋白；然而，它们在这些步骤中的作用机制仍然难以捉摸。在体内被三种解旋酶检查点阻断的剪接体被纯化，并对第一步阻断的剪接体进行了功能表征。我们表明，剪接的第一步需要一种新的 ATP 非依赖性构象变化。然后，Prp2p 催化一个 ATP 依赖性重排，将 SF3a 和 SF3b 复合物从剪接体中的分支点上置换下来。我们提出了一个模型，其中 SF3 在第一个转酯反应之前阻止分支点腺苷的化学活泼羟基的过早亲核攻击。当剪接体达到适当的构象并在 Prp2p 的作用下，SF3 从分支点上被置换下来，使得第一步化学反应得以发生。
