McCarthy Patrick, Garside Erin, Meschede-Krasa Yonatan, MacMillan Andrew, Pomeranz Krummel Daniel
Department of Biochemistry, Brandeis University, Waltham, MA, USA.
Department of Biochemistry, University of Alberta, Edmonton, Alberta, Canada.
Methods. 2017 Aug 1;125:25-35. doi: 10.1016/j.ymeth.2017.06.022. Epub 2017 Jun 23.
The spliceosome is a highly dynamic mega-Dalton enzyme, formed in part by assembly of U snRNPs onto its pre-mRNA substrate transcripts. Early steps in spliceosome assembly are challenging to study biochemically and structurally due to compositional and conformational dynamics. We detail an approach to covalently and reversibly constrain or trap non-covalent pre-mRNA/protein spliceosome complexes. This approach involves engineering a single disulfide bond between a thiol-bearing cysteine sidechain and a proximal backbone phosphate of the pre-mRNA, site-specifically modified with an N-thioalkyl moiety. When distance and angle between reactants is optimal, the sidechain will react with the single N-thioalkyl to form a crosslink upon oxidation. We provide protocols detailing how this has been applied successfully to trap an 11-subunit RNA-protein assembly, the human U1 snRNP, in complex with a pre-mRNA.
剪接体是一种高度动态的兆道尔顿酶，部分由U snRNP组装到其前体mRNA底物转录本上形成。由于组成和构象动力学，剪接体组装的早期步骤在生物化学和结构研究方面具有挑战性。我们详细介绍了一种共价且可逆地约束或捕获非共价前体mRNA/蛋白质剪接体复合物的方法。该方法涉及在带有硫醇的半胱氨酸侧链与经N-硫代烷基部分位点特异性修饰的前体mRNA的近端主链磷酸之间构建一个二硫键。当反应物之间的距离和角度最佳时，侧链将与单个N-硫代烷基反应，在氧化时形成交联。我们提供了详细的方案，说明如何成功应用此方法捕获一个由11个亚基组成的RNA-蛋白质组装体——人U1 snRNP，及其与前体mRNA的复合物。
