Mutant alleles of the MRS2 gene of yeast nuclear DNA suppress mutations in the catalytic core of a mitochondrial group II intron.
Mutant alleles of the MRS2 gene of yeast nuclear DNA suppress mutations in the catalytic core of a mitochondrial group II intron.
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酵母核 DNA 的 MRS2 基因的突变等位基因抑制线粒体 II 组内含子催化核心的突变。
DOI：
10.1006/jmbi.1998.2021
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发表时间：
1998
期刊：
影响因子：
--
通讯作者：
Perlman,PS
中科院分区：
文献类型：
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作者：
Schmidt,U;Maue,I;Lehmann,K;Belcher,SM;Stahl,U;Perlman,PS
先前的研究表明，一些酵母菌株携带的结构域5的点突变，阻止剪接的线粒体II组内含子产生自发的回复突变体，其中剪接部分恢复核基因的显性突变。在此，我们克隆并测序了一个这样的基因的抑制等位基因，并发现它是一个错义突变的MRS 2基因（MRS 2-L232 F）。MRS 2基因首先与II组内含子剪接有关，因为发现野生型基因的过表达微弱地抑制了另一个内含子突变的剪接缺陷。四分体分析表明，独立分离的两个其他结构域5突变的抑制子也是MRS 2基因的等位基因，DNA测序在每个菌株中鉴定出一个新的错义突变（MRS 2-T230 I和MRS 2-L213 M）。所有三个抑制基因突变导致温度敏感的呼吸缺陷，这是显性负杂合二倍体，但这些菌株剪接的突变内含子在高温下。这三个突变位于蛋白质的一个结构域中，该结构域可能是螺旋-转角-螺旋区域，因此突变对蛋白质-蛋白质相互作用的影响可能有助于这些表型。这些突变抑制了α I 5 γ的许多但不是全部可用剪接缺陷突变的剪接缺陷，包括几个内含子结构域的突变。蛋白质和RNA印迹实验表明，由MRS 2基因编码的蛋白质的水平，而不是mRNA，通过这些突变升高。有趣的是，野生型蛋白质的过表达恢复的剪接水平比突变Mrs 2蛋白质的类似升高水平所获得的剪接水平低得多。这些菌株的剪接表型表明Mrs 2蛋白对II组内含子剪接的直接作用，但尚未排除间接作用。
Previous studies show that some yeast strains carrying point mutations of domain 5 that block splicing of a mitochondrial group II intron yield spontaneous revertants in which splicing is partially restored by dominant mutations of nuclear genes. Here we cloned and sequenced the suppressor allele of one such gene, and found it to be a missense mutation of the MRS2 gene (MRS2-L232F). The MRS2 gene was first implicated in group II intron splicing by the finding that overexpression of the wild-type gene weakly suppresses the splicing defect of a mutation of another intron. Tetrad analysis showed that independently isolated suppressors of two other domain 5 mutations are also allelles of the MRS2 gene and DNA sequencing identified a new missense mutation in each strain (MRS2-T230I and MRS2-L213M). All three suppressor mutations cause a temperature-sensitive respiration defect that is dominant negative in heterozygous diploids, but those strains splice the mutant intron at the elevated temperature. The three mutations are in a domain of the protein that is likely to be a helix-turn-helix region, so that effects of the mutations on protein-protein interactions may contribute to these phenotypes. These mutations suppress the splicing defect of many, but not all, of the available splicing defective mutations of aI5γ, including mutations of several intron domains. Protein and RNA blot experiments show that the level of the protein encoded by the MRS2 gene, but not the mRNA, is elevated by these mutations. Interestingly, overexpression of the wild-type protein restores much lower levels of splicing than were obtained with similar elevated levels of the mutated Mrs2 proteins. The splicing phenotypes of these strains suggest a direct role for Mrs2 protein on group II intron splicing, but an indirect effect is not yet ruled out.