The effect on translational efficiency of the i6A37 modification in cytoplasmic and mitochondrial tRNAs: Mitochondrial and selenoproteins.

细胞质和线粒体 tRNA 中 i6A37 修饰对翻译效率的影响：线粒体和硒蛋白。

• 批准号: 277195918
• 负责人: Professor Dr. Ulrich Schweizer
• 金额: --
• 依托单位: Institut für Biochemie und Molekularbiologie (IBMB)
• 依托单位国家: 德国
• 项目类别: Priority Programmes
• 财政年份: 2015
• 资助国家: 德国
• 起止时间: 2014-12-31 至 2018-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/277195918?language=en
• 关键词: effect; translational; efficiency; i6A37; modification

To study how modified bases in the anticodon stem loop of tRNAs modulate translation of proteins, requires suitable models and methods that allow to measure translation at codon resolution. Ribosomal Profiling allows to measure translational efficiency of any codon on a genome-wide scale. N6-Isopentenylation of adenosine 37 (i6A37) occurs in several cytoplasmic and mitochondrial tRNAs. The only known enzyme responsible for tRNA isopentenylation is TRIT1 (MiaA in E. coli, MOD5 in S. cerevisiae) and only a single animal model of tRNA i6A-deficiency exists: the gro-1 mutant in C. elegans. Analysis of global effects on translation is inherently difficult, but we propose to focus on a clearly defined class of proteins which depend on translation by a single tRNA which reads only one codon: selenoproteins contain the rare amino acid selenocysteine (Sec), which is translated by its cognate tRNA[Ser]Sec. This tRNA reads UGA as Sec. The in-frame UGA occurs only once in each selenoprotein mRNA. It is known that among the 24 murine selenoproteins a hierarchy is established depending on the usage of differentially modified tRNA[Ser]Sec isoacceptors and interfering with i6A-modi-fication of tRNA[Ser]Sec reduced selenoprotein translation in a gene-specific pattern. We will study the direct influence of tRNASec isopentenylation on UGA re-coding in seleno¬proteins by Ribosomal Profiling in livers from our recently generated Trit1-deficient mice. Since some mitochondrial tRNAs also carry i6A, we will apply Ribosomal Profiling to the 13 mitochondrial-encoded proteins. Analysis of translation at the codon level of two clearly defined sets of proteins will reflect the effect of tRNA isopentenylation on translational efficiency, reading frame maintenance, and message stability in the specific sequence context of endogenous mRNAs. Very recently, a patient with a homozygous point mutation in TRIT1 has been identified with mitochondrial disease. Interestingly, initial analysis in our laboratory shows that selenoproteins are well expressed. This means that the mutation is TRIT1 selectively affects recognition of some substrate tRNAs. We thus want to study in vitro the substrate specificity of TRIT1 towards tRNAs by mutagenesis.

为了研究tRNA的反密码子茎环中修饰的碱基如何调节蛋白质的翻译，需要合适的模型和方法，以允许在密码子分辨率下测量翻译。核糖体分析允许在全基因组范围内测量任何密码子的翻译效率。腺苷37（i6 A37）的N6-异戊烯化发生在几种细胞质和线粒体tRNA中。唯一已知的负责tRNA异戊烯化的酶是TRIT 1（E. coli，S.酿酒酵母中的gro-1突变体，并且仅存在单一的tRNA i6 A缺陷的动物模型：优美的翻译的全球影响的分析是固有的困难，但我们建议集中在一个明确定义的蛋白质类，依赖于翻译由一个单一的tRNA，只读一个密码子：硒蛋白含有罕见的氨基酸硒代半胱氨酸（Sec），这是由它的同源tRNA[Ser]Sec翻译。该tRNA将UGA读作Sec。读框内UGA在每个硒蛋白mRNA中仅出现一次。已知在24种鼠硒蛋白中，依赖于差异修饰的tRNA[Ser]Sec同种受体的使用和干扰tRNA[Ser]Sec的i6 A修饰以基因特异性模式减少硒蛋白翻译，建立了等级。我们将在我们最近产生的Trit 1缺陷小鼠的肝脏中通过核糖体谱研究tRNASec异戊烯化对硒蛋白中UGA重编码的直接影响。由于一些线粒体tRNA也携带i6 A，我们将对13种线粒体编码的蛋白质进行核糖体分析。在密码子水平上分析两组明确定义的蛋白质的翻译将反映tRNA异戊烯化对内源性mRNA的特定序列背景下的翻译效率、阅读框架维持和信息稳定性的影响。最近，一位TRIT 1纯合子点突变的患者被确定患有线粒体疾病。有趣的是，我们实验室的初步分析表明，硒蛋白表达良好。这意味着TRIT 1突变选择性地影响某些底物tRNA的识别。因此，我们希望通过诱变在体外研究TRIT 1对tRNA的底物特异性。