EARLY COMMITMENT OF YEAST PRE-MESSENGER RNA TO THE SPLICEOSOME PATHWAY
EARLY COMMITMENT OF YEAST PRE-MESSENGER RNA TO THE SPLICEOSOME PATHWAY
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DOI：
10.1128/mcb.8.9.3755
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发表时间：
1988-09-01
影响因子：
5.3
通讯作者：
ROSBASH, M
中科院分区：
文献类型：
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作者：
LEGRAIN, P;SERAPHIN, B;ROSBASH, M
前体mRNA在体外剪接之前是复合物形成（剪接体组装）。U2小核RNA（snRNA）被发现在最早的形式的剪接体检测天然凝胶电泳，无论是在酿酒酵母和后生动物提取物。为了研究在酵母提取物中形成这种早期复合物（条带III）的要求，我们通过寡核苷酸指导的RNase H消化来切割U2 snRNA。U2 snRNA通过这种方式消耗抑制剪接和条带III形成。使用这种耗尽的提取物，我们能够设计一个追踪实验，该实验表明，前体mRNA底物致力于剪接体组装途径的功能性U2 snRNP的情况下。在定型步骤期间发生的相互作用对添加大量未标记底物具有高度抗性，并且需要很少或不需要ATP。已经通过缺失突变体的竞争实验分析了该定型步骤的序列要求：5“剪接位点共有序列和分支点TACTAAC盒序列都是必需的。这些实验强烈表明，初始组装过程需要反式作用因子（RNA和/或蛋白质），其在U2 snRNP结合之前识别并稳定结合前mRNA的两个共有序列。
Pre-mRNA splicing in vitro is preceded by complex formation (spliceosome assembly). U2 small nuclear RNA (snRNA) is found in the earliest form of the spliceosome detected by native gel electrophoresis, both in Saccharomyces cerevisiae and in metazoan extracts. To examine the requirements for the formation of this early complex (band III) in yeast extracts, we cleaved the U2 snRNA by oligonucleotide-directed RNase H digestion. U2 snRNA depletion by this means inhibits both splicing and band III formation. Using this depleted extract, we were able to design a chase experiment which shows that a pre-mRNA substrate is committed to the spliceosome assembly pathway in the absence of functional U2 snRNP. Interactions occurring during the commitment step are higly resistant to the addition of an excees of unlabeled substrate and require little or no ATP. Sequence requirements for this commitment step have been analyzed by competition experiments with deletion mutants: both the 5'' splice site consensus sequence and the branch point TACTAAC box sequence are necessary. These experiments strongly suggest that the initial assembly process requires a trans-acting factor(s) (RNA and/or proteins) that recognizes and stably binds to the two consensus sequences of the pre-mRNA prior to U2 snRNP binding.