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首页> 外文期刊>Molecular and Cellular Biology >Mutagenesis of SNM1, Which Encodes a Protein Component of the Yeast RNase MRP, Reveals a Role for This Ribonucleoprotein Endoribonuclease in Plasmid Segregation
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Mutagenesis of SNM1, Which Encodes a Protein Component of the Yeast RNase MRP, Reveals a Role for This Ribonucleoprotein Endoribonuclease in Plasmid Segregation

机译：编码酵母RNase MRP蛋白质成分的SNM1诱变揭示了这种核糖核酸内切核糖核酸酶在质粒分离中的作用

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RNase MRP is a ribonucleoprotein endoribonuclease that has been shown to have roles in both mitochondrial DNA replication and nuclear 5.8S rRNA processing. SNM1 encodes an essential 22.5-kDa protein that is a component of yeast RNase MRP. It is an RNA binding protein that binds the MRP RNA specifically. This 198-amino-acid protein can be divided into three structural regions: a potential leucine zipper near the amino terminus, a binuclear zinc cluster in the middle region, and a serine- and lysine-rich region near the carboxy terminus. We have performed PCR mutagenesis of the SNM1gene to produce 17 mutants that have a conditional phenotype for growth at different temperatures. Yeast strains carrying any of these mutations as the only copy of snm1 display an rRNA processing defect identical to that in MRP RNA mutants. We have characterized these mutant proteins for RNase MRP function by examining 5.8S rRNA processing, MRP RNA binding in vivo, and the stability of the RNase MRP RNA. The results indicate two separate functional domains of the protein, one responsible for binding the MRP RNA and a second that promotes substrate cleavage. The Snm1 protein appears not to be required for the stability of the MRP RNA, but very low levels of the protein are required for processing of the 5.8S rRNA. Surprisingly, a large number of conditional mutations that resulted from nonsense and frameshift mutations throughout the coding regions were identified. The most severe of these was a frameshift at amino acid 7. These mutations were found to be undergoing translational suppression, resulting in a small amount of full-length Snm1 protein. This small amount of Snm1 protein was sufficient to maintain enough RNase MRP activity to support viability. Translational suppression was accomplished in two ways. First, CEN plasmid missegregation leads to plasmid amplification, which in turn leads to SNM1 mRNA overexpression. Translational suppression of a small amount of the superabundant SNM1 mRNA results in sufficient Snm1 protein to support viability. CEN plasmid missegregation is believed to be the result of a prolonged telophase arrest that has been recently identified in RNase MRP mutants. Either the SNM1gene is inherently susceptible to translational suppression or extremely small amounts of Snm1 protein are sufficient to maintain essential levels of MRP activity.
机译：RNase MRP是一种核糖核蛋白内切核糖核酸酶，已被证明在线粒体DNA复制和核5.8S rRNA加工中均具有作用。 SNM1 编码一种必需的22.5-kDa蛋白，它是酵母RNase MRP的组成部分。它是一种RNA结合蛋白，可特异性结合MRP RNA。该198个氨基酸的蛋白质可分为三个结构区域：一个位于氨基末端附近的亮氨酸拉链，一个位于中部区域的双核锌簇以及一个位于羧基末端附近的富含丝氨酸和赖氨酸的区域。我们已经对 SNM1 基因进行了PCR诱变，以产生17个突变体，这些突变体具有在不同温度下生长的条件表型。携带任何这些突变的酵母菌株作为 snm1 的唯一副本显示出与MRP RNA突变体相同的rRNA加工缺陷。我们已经通过检查5.8S rRNA加工，体内MRP RNA结合以及RNase MRP RNA的稳定性来表征这些突变蛋白具有RNase MRP功能。结果表明蛋白质的两个独立的功能域，一个负责结合MRP RNA，第二个促进底物裂解。 Snm1蛋白似乎对于MRP RNA的稳定性不是必需的，但是加工5.8S rRNA所需的蛋白水平非常低。令人惊讶的是，鉴定了在整个编码区中由无义和移码突变引起的大量条件突变。其中最严重的是第7位氨基酸的移码。这些突变被发现受到翻译抑制，从而导致少量的全长Snm1蛋白。少量的Snm1蛋白足以维持足够的RNase MRP活性以支持生存能力。翻译抑制是通过两种方式完成的。首先， CEN 质粒错聚导致质粒扩增，继而导致 SNM1 mRNA过表达。少量过量的 SNM1 mRNA的翻译抑制可产生足够的Snm1蛋白来支持其生存能力。据信 CEN 质粒的失聚是最近在RNase MRP突变体中发现的延长的末期停滞的结果。 SNM1 基因天生就容易受到翻译抑制的影响，或者极少量的Snm1蛋白足以维持必需的MRP活性水平。

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