Using mitoribosomal profiling to investigate human mitochondrial translation.
Using mitoribosomal profiling to investigate human mitochondrial translation.
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DOI：
10.12688/wellcomeopenres.13119.2
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发表时间：
2017-01-01
影响因子：
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通讯作者：
Chrzanowska-Lightowlers, Zofia M A
中科院分区：
文献类型：
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作者：
Gao, Fei;Wesolowska, Maria;Chrzanowska-Lightowlers, Zofia M A
背景：人类线粒体中的基因表达具有多种特异性特征。其中之一是最近发现的前所未有的招聘的一个martinally-coded的tRNA作为一个结构组成部分的大线粒体亚基。在猪颗粒中，它是mt-tRNA Phe，而在人类中，它是mt-tRNA瓦尔。我们以前已经表明，当突变的mt-tRNA瓦尔导致非常低的稳态水平，有优先募集的mt-tRNA苯丙氨酸。我们已经研究了这种改变的线粒体是否会影响细胞器内蛋白质的合成。研究方法：通过使用线粒体分析，我们已经揭示了在正常条件下以及那些线粒体已纳入MT-tRNA苯丙氨酸的线粒体行为与其模板MT-mRNA的方面。结果如下：控制条件下的转录本上的线粒体驻留的分析表明，虽然线粒体仅使用22个MT-tRNA用于蛋白质合成，但在密码子位置3处使用非规范摆动碱基对不会引起任何可测量的差异，无论密码子如何。将异常的含有线粒体-tRNA苯丙氨酸的线粒体核糖体的概况与整合了线粒体-tRNA瓦尔的对照的概况进行比较，揭示了在后者中观察到的受损的翻译不是由于在编码这些氨基酸中的任一种的三联体上停滞。除了两个双顺反子RNA单元RNA 7和RNA 14中起始位点位于内部外，线粒体与起始密码子相互作用的改变不能直接归因于使用非同源起始密码子或5'前导序列的存在或不存在。结论：这些数据报告的权力，线粒体分析有助于了解哺乳动物线粒体蛋白质合成的微妙之处。对突变型mt-tRNA瓦尔细胞系的分析表明，尽管mt-tRNA Phe在猪线粒体中是优选的，但其整合到人类对应物中导致了次优结构，该结构改变了其与mt-mRNA的相互作用。
Background: Gene expression in human mitochondria has various idiosyncratic features. One of these was recently revealed as the unprecedented recruitment of a mitochondrially-encoded tRNA as a structural component of the large mitoribosomal subunit. In porcine particles this is mt-tRNA Phe whilst in humans it is mt-tRNA Val. We have previously shown that when a mutation in mt-tRNA Val causes very low steady state levels, there is preferential recruitment of mt-tRNA Phe. We have investigated whether this altered mitoribosome affects intra-organellar protein synthesis. Methods: By using mitoribosomal profiling we have revealed aspects of mitoribosome behaviour with its template mt-mRNA under both normal conditions as well as those where the mitoribosome has incorporated mt-tRNA Phe. Results: Analysis of the mitoribosome residency on transcripts under control conditions reveals that although mitochondria employ only 22 mt-tRNAs for protein synthesis, the use of non-canonical wobble base pairs at codon position 3 does not cause any measurable difference in mitoribosome occupancy irrespective of the codon. Comparison of the profile of aberrant mt-tRNA Phe containing mitoribosomes with those of controls that integrate mt-tRNA Val revealed that the impaired translation seen in the latter was not due to stalling on triplets encoding either of these amino acids. The alterations in mitoribosome interactions with start codons was not directly attributable to the either the use of non-cognate initiation codons or the presence or absence of 5' leader sequences, except in the two bicistronic RNA units, RNA7 and RNA14 where the initiation sites are internal. Conclusions: These data report the power of mitoribosomal profiling in helping to understand the subtleties of mammalian mitochondrial protein synthesis. Analysis of profiles from the mutant mt-tRNA Val cell line suggest that despite mt-tRNA Phe being preferred in the porcine mitoribosome, its integration into the human counterpart results in a suboptimal structure that modifies its interaction with mt-mRNAs.