Schmitt M E, Clayton D A
Department of Developmental Biology, Stanford University School of Medicine, California 94305-5427.
Mol Cell Biol. 1993 Dec;13(12):7935-41. doi: 10.1128/mcb.13.12.7935-7941.1993.
RNase MRP is a site-specific ribonucleoprotein endoribonuclease that cleaves RNA from the mitochondrial origin of replication in a manner consistent with a role in priming leading-strand DNA synthesis. Despite the fact that the only known RNA substrate for this enzyme is complementary to mitochondrial DNA, the majority of the RNase MRP activity in a cell is found in the nucleus. The recent characterization of this activity in Saccharomyces cerevisiae and subsequent cloning of the gene coding for the RNA subunit of the yeast enzyme have enabled a genetic approach to the identification of a nuclear role for this ribonuclease. Since the gene for the RNA component of RNase MRP, NME1, is essential in yeast cells and RNase MRP in mammalian cells appears to be localized to nucleoli within the nucleus, we utilized both regulated expression and temperature-conditional mutations of NME1 to assay for a possible effect on rRNA processing. Depletion of the RNA component of the enzyme was accomplished by using the glucose-repressed GAL1 promoter. Shortly after the shift to glucose, the RNA component of the enzyme was found to be depleted severely, and rRNA processing was found to be normal at all sites except the B1 processing site. The B1 site, at the 5' end of the mature 5.8S rRNA, is actually composed of two cleavage sites 7 nucleotides apart. This cleavage normally generates two species of 5.8S rRNA at a ratio of 10:1 (small to large) in most eukaryotes. After RNase MRP depletion, yeast cells were found to have almost exclusively the larger species of 5.8S rRNA. In addition, an aberrant 309-nucleotide precursor that stretched from the A2 to E processing sites of rRNA accumulated in these cells. Temperature-conditional mutations in the RNase MRP RNA gene gave an identical phenotype. Translation in yeast cells depleted of the smaller 5.8S rRNA was found to remain robust, suggesting a possible function for two 5.8S rRNAs in the regulated translation of select messages. These results are consistent with RNase MRP playing a role in a late step of rRNA processing. The data also indicate a requirement for having the smaller form of 5.8S rRNA, and they argue for processing at the B1 position being composed of two separate cleavage events catalyzed by two different activities.
核糖核酸酶MRP是一种位点特异性核糖核蛋白内切核糖核酸酶，它以一种与引发前导链DNA合成作用一致的方式切割来自线粒体复制起点的RNA。尽管该酶唯一已知的RNA底物与线粒体DNA互补，但细胞中大部分核糖核酸酶MRP活性却存在于细胞核中。最近对酿酒酵母中这种活性的表征以及随后对酵母酶RNA亚基编码基因的克隆，使得能够采用遗传学方法来确定这种核糖核酸酶在细胞核中的作用。由于核糖核酸酶MRP的RNA组分基因NME1在酵母细胞中是必需的，并且哺乳动物细胞中的核糖核酸酶MRP似乎定位于细胞核内的核仁，我们利用NME1的调控表达和温度条件突变来检测其对rRNA加工可能产生的影响。通过使用葡萄糖抑制的GAL1启动子来实现该酶RNA组分的消耗。在转换到葡萄糖后不久，发现该酶的RNA组分严重减少，并且除了B1加工位点外，所有位点的rRNA加工均正常。B1位点位于成熟5.8S rRNA的5'端，实际上由两个相隔7个核苷酸的切割位点组成。在大多数真核生物中，这种切割通常以10:1（小到大）的比例产生两种5.8S rRNA。在核糖核酸酶MRP消耗后，发现酵母细胞几乎只含有较大的5.8S rRNA种类。此外，一种从rRNA的A2到E加工位点延伸的异常309核苷酸前体在这些细胞中积累。核糖核酸酶MRP RNA基因中的温度条件突变产生了相同的表型。发现在缺乏较小5.8S rRNA的酵母细胞中的翻译仍然强劲，这表明两种5.8S rRNA在特定信息的调控翻译中可能具有功能。这些结果与核糖核酸酶MRP在rRNA加工的后期步骤中起作用一致。数据还表明需要有较小形式的5.8S rRNA，并且它们支持在B1位置的加工由两种不同活性催化的两个独立切割事件组成。
