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

为了研究trna的反密码子干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来测量密码子分辨率的翻译。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 （i6A37）的n6 -异戊烯化发生在一些细胞质和线粒体trna中。唯一已知的负责tRNA异戊烯化的酶是TRIT1（大肠杆菌中的MiaA，酿酒葡萄球菌中的MOD5）， tRNA i6a缺乏的动物模型只有一种：秀丽隐杆线虫中的gro1突变体。分析对翻译的整体影响本身就很困难，但我们建议将重点放在一类明确定义的蛋白质上，这些蛋白质依赖于只读取一个密码子的单个tRNA的翻译：硒蛋白含有罕见的氨基酸硒半胱氨酸（Sec），它由其同源tRNA[Ser]Sec翻译。该tRNA将UGA读取为Sec。帧内UGA在每个硒蛋白mRNA中只发生一次。众所周知，在24种小鼠硒蛋白中，一个层次结构的建立取决于差异修饰tRNA[Ser]Sec同受体的使用，干扰i6a修饰tRNA[Ser]Sec会以基因特异性模式减少硒蛋白的翻译。我们将研究tRNASec异戊烯化对硒蛋白中UGA重编码的直接影响，通过对我们新生成的trit1缺陷小鼠肝脏的核糖体分析。由于一些线粒体trna也携带i6A，我们将对13种线粒体编码的蛋白质应用核糖体分析。分析两组明确定义的蛋白质在密码子水平上的翻译，将反映tRNA异戊烯化在内源性mrna特定序列背景下对翻译效率、阅读框维持和信息稳定性的影响。最近，一名患有TRIT1纯合点突变的患者被确认患有线粒体疾病。有趣的是，我们实验室的初步分析表明硒蛋白表达良好。这意味着突变是TRIT1选择性地影响一些底物trna的识别。因此，我们想在体外通过诱变研究TRIT1对trna的底物特异性。

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.