SPLICING OF YEAST NUCLEAR PRE-MESSENGER-RNA INVITRO REQUIRES A FUNCTIONAL 40S SPLICEOSOME AND SEVERAL EXTRINSIC FACTORS
SPLICING OF YEAST NUCLEAR PRE-MESSENGER-RNA INVITRO REQUIRES A FUNCTIONAL 40S SPLICEOSOME AND SEVERAL EXTRINSIC FACTORS
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DOI：
10.1101/gad.1.1.7
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发表时间：
1987-03-01
影响因子：
10.5
通讯作者：
ABELSON, J
中科院分区：
文献类型：
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作者：
LIN, RJ;LUSTIG, AJ;ABELSON, J
我们之前已经证明，大多数酵母温度敏感RNA突变体制备的提取物对体外前mRNA剪接是热敏感的，并且相应RNA基因的产物对于剪接区域的早期阶段是必需的。在本报告中，我们证明大多数热灭活突变体提取物不会形成剪接体，这表明它们的基因产物可能参与剪接体的形成。另一方面，热灭活的 rna2 提取物确实形成了仅含有未切割的前 mRNA 的剪接依赖性 40S 复合物。在 ATP 和补充提取物存在的情况下，40S 复合物中的前 mRNA 可以转化为剪接产物。这些结果表明：（1）热灭活rna2提取物中形成的40S复合物是剪接体（称为rna2.DELTA.剪接体），（2）剪接体是剪接途径中的功能中间体，（3）剪接过程可以分为两个步骤，剪接体形成和切割连接反应。其他结果表明，至少需要两个外在因子以及 RNA2 基因产物来补充 rna2.DELTA。剪接体。提出了酵母核前 mRNA 剪接的三步机制。
We have previously shown that extracts prepared from most of the yeast temperature-sensitive rna mutants are heat sensitive for pre-mRNA splicing in vitro, and that the products of the corresponding RNA genes are essential for the early stages of the splicing region. In this report, we demonstrate that most heat-inactivated mutant extracts do not form the spliceosome, suggesting, that their gene products are likely to be involved in spliceosome formation. Heat-inactivated rna2 extracts, on the other hand, do form a splicing-dependent 40S complex containing uncleaved pre-mRNA exclusively. The pre-mRNA in the 40S complex can be converted to the splicing products in the presence of ATP and complementing extracts. These results demonstrate that: (1) the 40S complex formed in heat-inactivated rna2 extracts is a spliceosome (termed the rna2.DELTA. spliceosome), (2) the spliceosome is a functional intermediate in the splicing pathway, and (3) the splicing process can be dissected into two steps, spliceosome formation and cleavage-ligation reactions. Additional results indicate that at least two extrinsic factors, as well as the RNA2 gene product, are required for complementation of the rna2.DELTA. spliicesome. A three-step mechanism for nuclear pre-mRNA splicing in yeast is proposed.