Assembly and dynamics of the U4/U6 di-snRNP by single-molecule FRET.
Assembly and dynamics of the U4/U6 di-snRNP by single-molecule FRET.
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DOI：
10.1093/nar/gkv1011
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发表时间：
2015-12-15
影响因子：
14.9
通讯作者：
Rueda D
中科院分区：
文献类型：
--
作者：
Hardin JW;Warnasooriya C;Kondo Y;Nagai K;Rueda D
在大型核糖核蛋白机器（例如核糖体和剪接体）中，RNA 充当组装支架以及关键的催化成分。蛋白质与 RNA 支架的结合可以诱导结构变化，进而调节其他成分的后续结合。剪接体 U4/U6 di-snRNP 包含广泛碱基配对的 U4 和 U6 snRNA、Snu13、Prp31、Prp3 和 Prp4、7 个 Sm 和 7 个 LSm 蛋白。我们通过电泳迁移率变动分析研究了 di-snRNP 组装过程中所有蛋白质成分与 snRNA 双链体的连续结合，并通过单分子 FRET 研究了 U4/U6 RNA 3 路连接中伴随的构象变化。发现双链体的茎 I 和 II 在游离 RNA 中同轴堆叠，并在整个组装过程中充当刚性支架，但 U4 snRNA 5' 茎环采用替代方向，每个方向均由 Prp31 和 Prp3/4 结合稳定，解释了 Prp31 存在下改变的 Prp3/4 结合亲和力。
In large ribonucleoprotein machines, such as ribosomes and spliceosomes, RNA functions as an assembly scaffold as well as a critical catalytic component. Protein binding to the RNA scaffold can induce structural changes, which in turn modulate subsequent binding of other components. The spliceosomal U4/U6 di-snRNP contains extensively base paired U4 and U6 snRNAs, Snu13, Prp31, Prp3 and Prp4, seven Sm and seven LSm proteins. We have studied successive binding of all protein components to the snRNA duplex during di-snRNP assembly by electrophoretic mobility shift assay and accompanying conformational changes in the U4/U6 RNA 3-way junction by single-molecule FRET. Stems I and II of the duplex were found to co-axially stack in free RNA and function as a rigid scaffold during the entire assembly, but the U4 snRNA 5′ stem-loop adopts alternative orientations each stabilized by Prp31 and Prp3/4 binding accounting for altered Prp3/4 binding affinities in presence of Prp31.