Ilagan Janine O, Jurica Melissa S
Department of Molecular, Cell and Developmental Biology and Center for Molecular Biology of RNA, University of California, Santa Cruz, CA, USA.
Methods Mol Biol. 2014;1126:179-92. doi: 10.1007/978-1-62703-980-2_14.
Isolating spliceosomes at a specific assembly stage requires a means to stall or enrich for one of the intermediate splicing complexes. We describe strategies to arrest spliceosomes at different points of complex formation and provide a detailed protocol developed for isolating intact splicing complexes arrested between the first and second chemical steps of splicing. Briefly, spliceosomes are assembled on a radiolabeled in vitro-transcribed splicing substrate from components present in nuclear extract of HeLa cells. Spliceosome progression is arrested after the first step of splicing chemistry by mutating the pre-mRNA substrate at the 3' splice site. The substrate also contains binding sites for the MS2 protein, which serve as an affinity tag. Purification of arrested spliceosomes is carried out in two steps: (1) size exclusion chromatography and (2) affinity selection via a fusion of MS2 and maltose-binding protein (MBP). Complex assembly and purification are analyzed by denaturing polyacrylamide gel electrophoresis.
在特定组装阶段分离剪接体需要一种使中间剪接复合物之一停滞或富集的方法。我们描述了在复合物形成的不同点捕获剪接体的策略，并提供了一个详细的方案，该方案是为分离在剪接的第一步和第二步化学步骤之间停滞的完整剪接复合物而制定的。简而言之，剪接体是从HeLa细胞核提取物中的成分在放射性标记的体外转录剪接底物上组装而成的。通过在3'剪接位点突变前体mRNA底物，剪接体在剪接化学的第一步之后停滞。该底物还包含MS2蛋白的结合位点，其作为亲和标签。停滞的剪接体的纯化分两步进行：（1）尺寸排阻色谱法和（2）通过MS2和麦芽糖结合蛋白（MBP）的融合进行亲和选择。通过变性聚丙烯酰胺凝胶电泳分析复合物的组装和纯化。
