Structuring of the 3′ splice site by U2AF65
Structuring of the 3′ splice site by U2AF65
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DOI：
10.1074/jbc.m307976200
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发表时间：
2003-12-12
影响因子：
4.8
通讯作者：
MacMillan, AM
中科院分区：
文献类型：
--
作者：
Kent, OA;Reayi, A;MacMillan, AM
哺乳动物内含子中 3' 剪接位点的识别是通过剪接因子 U2AF 与多蛋白剪接定型复合物中的前体 mRNA (pre-mRNA) 的关联来完成的。众所周知，这种相互作用涉及大 U2AF65 亚基与 3' 剪接位点上游序列的结合，但该蛋白质的四个结构域相对于 RNA 的方向以及它们在构建定型复合物中的作用仍不清楚，并且是矛盾模型的基础。我们使用一系列在N末端用定向羟基自由基探针铁-EDTA修饰的U2AF缺失突变体检查了U2AF65与代表3'剪接位点的RNA的相互作用。这些研究结合了对现存蛋白质高分辨率 X 射线结构的分析。 RNA复合物提出了一种模型，通过该模型，U2AF65弯曲前mRNA以并置前mRNA底物的反应功能，并组织这些结构以用于随后的剪接体组装。
Recognition of the 3 ' splice site in mammalian introns is accomplished by association of the splicing factor U2AF with the precursor mRNA ( pre- mRNA) in a multiprotein splicing commitment complex. It is well established that this interaction involves binding of the large U2AF65 subunit to sequences upstream of the 3 ' splice site, but the orientation of the four domains of this protein with respect to the RNA and hence their role in structuring the commitment complex remain unclear and the basis of contradictory models. We have examined the interaction of U2AF65 with an RNA representing the 3 ' splice site using a series of U2AF deletion mutants modified at the N terminus with the directed hydroxyl radical probe iron- EDTA. These studies, combined with an analysis of extant high resolution x- ray structures of protein . RNA complexes, suggest a model whereby U2AF65 bends the pre- mRNA to juxtapose reactive functionalities of the pre- mRNA substrate and organize these structures for subsequent spliceosome assembly.