Assembly and glycerol gradient isolation of yeast spliceosomes containing transcribed or synthetic U6 snRNA.
Assembly and glycerol gradient isolation of yeast spliceosomes containing transcribed or synthetic U6 snRNA.
复制标题
含有转录或合成的 U6 snRNA 的酵母剪接体的组装和甘油梯度分离。
DOI：
10.1007/978-1-60327-475-3_4
复制
发表时间：
2008
期刊：
影响因子：
--
通讯作者：
Lin,Ren-Jang
中科院分区：
文献类型：
--
作者：
Dery,KennethJ;Yean,Shyue-Lee;Lin,Ren-Jang
RNA-蛋白质相互作用的研究通常需要使用体外合成的RNA或重组蛋白组装RNA-蛋白质复合物。在这里，我们描述了一种使用转录或合成的RNA在酵母提取物中组装功能性剪接体的方案。体外组装的剪接体是稳定的，并且可以通过甘油梯度沉降分离用于后续分析。该方案描述了两种制备RNA的方法：使用噬菌体RNA聚合酶或使用T4 DNA连接酶连接RNA寡核苷酸。我们还描述了剪接感受态酵母提取物的制备，剪接体的组装，以及通过甘油梯度沉降分离剪接体。为了允许外源添加的U6 RNA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消除;然后添加“中和”U6 DNA寡核苷酸以保护进入的U6 RNA。该方案允许研究单个碱基或磷酸骨架的U6在剪接中的作用以及U6 snRNA和剪接体蛋白之间的相互作用。
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 bacte-riophage 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.