Mutations in conserved intron sequences affect multiple steps in the yeast splicing pathway, particularly assembly of the spliceosome.
Mutations in conserved intron sequences affect multiple steps in the yeast splicing pathway, particularly assembly of the spliceosome.
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保守内含子序列的突变影响酵母剪接途径中的多个步骤，特别是剪接体的组装。
DOI：
10.1002/j.1460-2075.1986.tb04412.x
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发表时间：
1986
期刊：
影响因子：
--
通讯作者：
Guthrie,C
中科院分区：
文献类型：
--
作者：
Vijayraghavan,U;Parker,R;Tamm,J;Iimura,Y;Rossi,J;Abelson,J;Guthrie,C
酵母内含子含有三个高度保守的序列，已知它们是前mRNA剪接所需的。使用体外诱变，我们已经合成了7个点突变在5个不同的位点，这些信号在酵母肌动蛋白内含子。然后将突变的内含子插入到三个构建体中的每一个中，这使我们能够评估体内和体外的结果。几乎在每种情况下，我们发现剪接的效率都被显著抑制;体内成熟mRNA水平范围为野生型的0 - 47%。令人惊讶的是，最紧密的突变不一定在溶核切割和分支形成的位点;因此这些核苷酸是高度优选的，但不是绝对必需的。此外，虽然特定的核苷酸是剪接的最后一步，即3′切割和外显子连接所特别需要的，但保守信号内突变的主要结果似乎是抑制剪接复合物的组装。
Yeast introns contain three highly conserved sequences which are known to be required for splicing of pre‐mRNA. Using in vitro mutagenesis, we have synthesized seven point mutations at five different sites in these signals in the yeast actin intron. The mutant introns were then inserted into each of three constructs, which allowed us to assess the consequences both in vivo and in vitro. In virtually every case, we found the efficiency of splicing to be significantly depressed; mature mRNA levels in vivo ranged from 0 to 47% of wild‐type. Surprisingly, the tightest mutations were not necessarily at the sites of nucleolytic cleavage and branch formation; these nucleotides are thus highly preferred, but are not absolutely necessary. Moreover, while particular nucleotides are specifically required for the final step in splicing, i.e. 3′ cleavage and exon ligation, the predominant consequence of mutation within the conserved signals appears to be the inhibition of assembly of the splicing complex.