Department of Biochemistry and Biophysics, University of Rochester School of Medicine and Dentistry, Rochester, NY 14642, USA.
J Mol Biol. 2011 Feb 4;405(5):1128-38. doi: 10.1016/j.jmb.2010.11.054. Epub 2010 Dec 10.
Spliceosomes assemble on pre-mRNA splice sites through a series of dynamic ribonucleoprotein complexes, yet the nature of the conformational changes remains unclear. Splicing factor 1 (SF1) and U2 auxiliary factor (U2AF(65)) cooperatively recognize the 3' splice site during the initial stages of pre-mRNA splicing. Here, we used small-angle X-ray scattering to compare the molecular dimensions and ab initio shape restorations of SF1 and U2AF(65) splicing factors, as well as the SF1/U2AF(65) complex in the absence and presence of AdML (adenovirus major late) splice site RNAs. The molecular dimensions of the SF1/U2AF(65)/RNA complex substantially contracted by 15 Å in the maximum dimension, relative to the SF1/U2AF(65) complex in the absence of RNA ligand. In contrast, no detectable changes were observed for the isolated SF1 and U2AF(65) splicing factors or their individual complexes with RNA, although slight differences in the shapes of their molecular envelopes were apparent. We propose that the conformational changes that are induced by assembly of the SF1/U2AF(65)/RNA complex serve to position the pre-mRNA splice site optimally for subsequent stages of splicing.
剪接体通过一系列动态的核糖核蛋白复合物在 pre-mRNA 剪接位点上组装，但其构象变化的性质仍不清楚。剪接因子 1 (SF1) 和 U2 辅助因子 (U2AF(65)) 在 pre-mRNA 剪接的初始阶段协同识别 3' 剪接位点。在这里，我们使用小角 X 射线散射来比较 SF1 和 U2AF(65) 剪接因子以及 SF1/U2AF(65) 复合物在不存在和存在 AdML（腺病毒主要晚期）剪接位点 RNA 时的分子尺寸和从头形状恢复。与没有 RNA 配体的 SF1/U2AF(65) 复合物相比，SF1/U2AF(65)/RNA 复合物的最大尺寸收缩了 15 Å，分子尺寸显著收缩。相比之下，对于分离的 SF1 和 U2AF(65) 剪接因子或它们与 RNA 的单个复合物，没有观察到可检测到的变化，尽管它们的分子包络形状略有不同。我们提出，SF1/U2AF(65)/RNA 复合物组装所诱导的构象变化有助于将 pre-mRNA 剪接位点最佳定位，以进行后续的剪接阶段。
