Yeast mitochondrial protein Pet111p binds directly to two distinct targets in COX2 mRNA, suggesting a mechanism of translational activation
Yeast mitochondrial protein Pet111p binds directly to two distinct targets in COX2 mRNA, suggesting a mechanism of translational activation
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DOI：
10.1074/jbc.ra118.005355
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发表时间：
2019-05-03
影响因子：
4.8
通讯作者：
Anikin, Michael
中科院分区：
文献类型：
--
作者：
Jones, Julia L.;Hofmann, Katharina B.;Anikin, Michael
线粒体DNA中的基因编码呼吸链复合体的必需亚基。在酵母中，线粒体基因的表达由一组在细胞核中编码的基因特异性翻译激活因子控制。这些因素似乎是调节系统的一部分，该系统能够协调表达来自核和线粒体基因组的必要基因，以产生功能性呼吸复合物。许多翻译激活剂被认为作用于靶mRNA的5-非翻译区，但参与这种调节的分子机制仍然不清楚。在这项研究中，我们使用了体内和体外分析相结合的特点，这些翻译激活剂之一，pentatricopeptide重复蛋白Pet 111 p，其推定的目标，COX 2 mRNA，编码亚基II的细胞色素c氧化酶的相互作用。使用光活化核糖核苷增强的交联和免疫沉淀分析，我们发现Pet 111 p直接和特异性结合到COX 2转录本的5端近端区域。此外，我们应用体外RNA酶足迹法，并映射了蛋白质的两个结合靶点，其中一个位于5-非翻译前导序列中，另一个位于编码序列内。结合现有的遗传数据，这些结果表明一个合理的翻译激活机制，其中Pet 111 p的结合可能会阻止抑制性二级结构形成的翻译起始区，从而使mRNA可用于与核糖体的相互作用。
The genes in mitochondrial DNA code for essential subunits of the respiratory chain complexes. In yeast, expression of mitochondrial genes is controlled by a group of gene-specific translational activators encoded in the nucleus. These factors appear to be part of a regulatory system that enables concerted expression of the necessary genes from both nuclear and mitochondrial genomes to produce functional respiratory complexes. Many of the translational activators are believed to act on the 5-untranslated regions of target mRNAs, but the molecular mechanisms involved in this regulation remain obscure. In this study, we used a combination of in vivo and in vitro analyses to characterize the interactions of one of these translational activators, the pentatricopeptide repeat protein Pet111p, with its presumed target, COX2 mRNA, which encodes subunit II of cytochrome c oxidase. Using photoactivatable ribonucleoside-enhanced cross-linking and immunoprecipitation analysis, we found that Pet111p binds directly and specifically to a 5-end proximal region of the COX2 transcript. Further, we applied in vitro RNase footprinting and mapped two binding targets of the protein, of which one is located in the 5-untranslated leader and the other is within the coding sequence. Combined with the available genetic data, these results suggest a plausible mechanism of translational activation, in which binding of Pet111p may prevent inhibitory secondary structures from forming in the translation initiation region, thus rendering the mRNA available for interaction with the ribosome.