Department of Biochemistry, School of Molecular and Systems Medicine, University of Alberta, Edmonton, Alberta, Canada.
RNA. 2011 Jan;17(1):155-65. doi: 10.1261/rna.2224411. Epub 2010 Nov 9.
Human p14 (SF3b14), a component of the spliceosomal U2 snRNP, interacts directly with the pre-mRNA branch adenosine within the context of the bulged duplex formed between the pre-mRNA branch region and U2 snRNA. This association occurs early in spliceosome assembly and persists within the fully assembled spliceosome. Analysis of the crystal structure of a complex containing p14 and a peptide derived from p14-associated SF3b155 combined with the results of cross-linking studies has suggested that the branch nucleotide interacts with a pocket on a non-canonical RNA binding surface formed by the complex. Here we report a structural model of the p14 · bulged duplex interaction based on a combination of X-ray crystallography of an adenine p14/SF3b155 peptide complex, biochemical comparison of a panel of disulfide cross-linked protein-RNA complexes, and small-angle X-ray scattering (SAXS). These studies reveal specific recognition of the branch adenosine within the p14 pocket and establish the orientation of the bulged duplex RNA bound on the protein surface. The intimate association of one surface of the bulged duplex with the p14/SF3b155 peptide complex described by this model buries the branch nucleotide at the interface and suggests that p14 · duplex interaction must be disrupted before the first step of splicing.
人类 p14（SF3b14）是剪接体 U2 snRNP 的一个组成部分，它直接与前体 mRNA 分支腺苷相互作用，而该腺苷位于前体 mRNA 分支区域与 U2 snRNA 之间形成的凸起双链体的背景下。这种结合发生在剪接体组装的早期，并在完全组装的剪接体中持续存在。对包含 p14 和源自与 p14 相关的 SF3b155 的肽的复合物的晶体结构的分析，以及交联研究的结果，表明分支核苷酸与由复合物形成的非典型 RNA 结合表面上的口袋相互作用。在这里，我们报告了基于腺嘌呤 p14/SF3b155 肽复合物的 X 射线晶体学、一系列二硫键交联蛋白-RNA 复合物的生化比较以及小角 X 射线散射 (SAXS) 的组合，对 p14·凸起双链体相互作用的结构模型。这些研究揭示了 p14 口袋内分支腺苷的特异性识别，并确定了结合在蛋白质表面上的凸起双链 RNA 的取向。该模型描述的凸起双链体与 p14/SF3b155 肽复合物的一个表面的紧密结合将分支核苷酸埋藏在界面处，并表明 p14·双链体相互作用必须在剪接的第一步之前被破坏。
