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

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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.