Dery Kenneth J, Yean Shyue-Lee, Lin Ren-Jang
Beckman Research Institute of the City of Hope, Duarte, California, USA.
Methods Mol Biol. 2008;488:41-63. doi: 10.1007/978-1-60327-475-3_4.
Studies of RNA-protein interactions often require assembly of the RNA-protein complex using in vitro synthesized RNA or recombinant protein. Here, we describe a protocol to assemble a functional spliceosome in yeast extracts using transcribed or synthetic RNAs. The in vitro assembled spliceosome is stable and can be isolated by sedimentation through glycerol gradients for subsequent analysis. The protocols describe two procedures to prepare RNA: using bacteriophage RNA polymerases or ligation of RNA oligos using T4 DNA ligase. We also describe the preparation of splicing competent yeast extracts, the assembly of the spliceosome, and the isolation of the spliceosome by glycerol gradient sedimentation. To allow exogenously added U6 RNA to be incorporated into the spliceosome, the endogenous U6 small nuclear RNA (snRNA) in the extract is eliminated by an antisense U6 DNA oligo and ribonuclease H; a "neutralizing" U6 DNA oligo was then added to protect the incoming U6 RNA. This protocol allows study of the role individual bases or the phosphate backbone of U6 plays in splicing and of the interaction between U6 snRNA and the spliceosomal proteins.
对RNA-蛋白质相互作用的研究通常需要使用体外合成的RNA或重组蛋白来组装RNA-蛋白质复合物。在此，我们描述了一种在酵母提取物中使用转录的或合成的RNA组装功能性剪接体的方法。体外组装的剪接体是稳定的，可通过甘油梯度沉降进行分离，以便后续分析。该方法描述了两种制备RNA的程序：使用噬菌体RNA聚合酶或使用T4 DNA连接酶连接RNA寡核苷酸。我们还描述了具有剪接活性的酵母提取物的制备、剪接体的组装以及通过甘油梯度沉降分离剪接体。为了使外源添加的U6 RNA能够掺入剪接体中，提取物中的内源性U6小核RNA（snRNA）通过反义U6 DNA寡核苷酸和核糖核酸酶H被去除；然后添加一种“中和”U6 DNA寡核苷酸以保护导入的U6 RNA。该方法允许研究U6的单个碱基或磷酸骨架在剪接中的作用以及U6 snRNA与剪接体蛋白之间的相互作用。
