RNA structure analysis of human spliceosomes reveals a compact 3D arrangement of snRNAs at the catalytic core
RNA structure analysis of human spliceosomes reveals a compact 3D arrangement of snRNAs at the catalytic core
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DOI：
10.1038/emboj.2013.198
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发表时间：
2013-10-30
期刊：
影响因子：
11.4
通讯作者：
Luehrmann, Reinhard
中科院分区：
文献类型：
--
作者：
Anokhina, Maria;Bessonov, Sergey;Luehrmann, Reinhard
尽管U snRNA在剪接中发挥着重要作用，但人们对U2、U6和U 5 snRNA以及活性剪接体中前mRNA的3D排列知之甚少。为了阐明它们的相对空间组织和动态重排，我们研究了亲和纯化的人剪接体的RNA结构之前和之后的催化步骤1通过化学RNA结构探测。我们发现，在活性剪接体中，U2/U6 snRNA双链体的稳定3向连接在步骤1中保持最低程度。此外，在人剪接体复合物的不同组装阶段（即B、B act或C复合物）中未检测到在酵母中观察到的交替、互斥的U2 snRNA构象的形成。Peglien交联揭示了U 5 snRNA的内环1和紧邻分支点下游的内含子核苷酸之间在步骤1期间/之后的相互作用。使用实验得出的结构约束，我们产生了一个模型的RNA网络的步骤1剪接体，基于晶体结构的第II组内含子通过同源性建模。该模型与当前的遗传、生化和结构数据在拓扑上是一致的。
Although U snRNAs play essential roles in splicing, little is known about the 3D arrangement of U2, U6, and U5 snRNAs and the pre-mRNA in active spliceosomes. To elucidate their relative spatial organization and dynamic rearrangement, we examined the RNA structure of affinity-purified, human spliceosomes before and after catalytic step 1 by chemical RNA structure probing. We found a stable 3-way junction of the U2/U6 snRNA duplex in active spliceosomes that persists minimally through step 1. Moreover, the formation of alternating, mutually exclusive, U2 snRNA conformations, as observed in yeast, was not detected in different assembly stages of human spliceosomal complexes (that is, B, B act, or C complexes). Psoralen crosslinking revealed an interaction during/after step 1 between internal loop 1 of the U5 snRNA, and intron nucleotides immediately downstream of the branchpoint. Using the experimentally derived structural constraints, we generated a model of the RNA network of the step 1 spliceosome, based on the crystal structure of a group II intron through homology modelling. The model is topologically consistent with current genetic, biochemical, and structural data.