Lack of tRNA-i6A modification causes mitochondrial-like metabolic deficiency in S. pombe by limiting activity of cytosolic tRNA^Tyr, not mito-tRNA

Abstract

tRNA-isopentenyl transferases (IPTases) are highly conserved enzymes that form isopentenyl-N 6 -A37 (i6A37) on subsets of tRNAs, enhancing their translation activity. Nuclear-encoded IPTases modify select cytosolic (cy-) and mitochondrial (mt-) tRNAs. Mutation in human IPTase, TRIT1, causes disease phenotypes characteristic of mitochondrial translation deficiency due to mt-tRNA dysfunction. Deletion of the Schizosaccharomyces pombe IPTase (tit1-Δ) causes slow growth in glycerol, as well as in rapamycin, an inhibitor of TOR kinase that maintains metabolic homeostasis. . We show that lower ATP levels in tit1-Δ relative to tit1 + cells are also more decreased by an inhibitor of oxidative phosphorylation, indicative of mitochondrial dysfunction. Here we asked if the tit1-Δ phenotypes are due to hypomodification of cy-tRNA or mt-tRNA. A cytosol-specific IPTase that modifies cy-tRNA, but not mt-tRNA, fully rescues the tit1-Δ phenotypes. Moreover, overexpression of cy-tRNAs also rescues the phenotypes, and cy-tRNA Tyr alone substantially does so. Bioinformatics indicate that cy-tRNA Tyr is most limiting for codon demand in tit1-Δ cells and that the cytosolic mRNAs most loaded with Tyr codons encode carbon metabolilizing enzymes, many of which are known to localize to mitochondria. Thus, S. pombe i6A37 hypomodificationassociated metabolic deficiency results from hypoactivity of cy-tRNA, mostly tRNA Tyr , and unlike human TRIT1-deficiency does not impair mitochondrial translation due to mt-tRNA hypomodification. We discuss species-specific aspects of i6A37. Specifically relevant to mitochondria, we show that its hypermodified version, ms2i6A37 (2-methylthiolated), which occurs on certain mammalian mt-tRNAs (but not cy-tRNAs), is not found in yeast.

Evolving specificity of tRNA 3-methyl-cytidine-32 (m3C32) modification: a subset of tRNAsSer requires N6-isopentenylation of A37

“…Finally, we note that the findings reported here may be relevant to a heritable disorder caused by mutation of human tRNA isopentenyl transferase, TRIT1 and associated i 6 A37 hypomodification of cytoplasmic and mitochondrial tRNA (Yarham et al 2014;Lamichhane et al 2016). The data suggest that human i 6 A37 hypomodification may be accompanied by m 1998).…”

Factors That Shape Eukaryotic tRNAomes: Processing, Modification and Anticodon–Codon Use

“…tRNA Ser IGA must decode UCU and UCC codons as there is no tRNA-G34 anticodon (G34 anticodon-sparing) in the S. pombe tRNAome. The UCU codon is used 2.5-fold more frequently than the UCC codon in the overall S. pombe transcriptome [20], but the ratios differ four-fold in high vs. low-expression mRNAs ([102] and references therein). As the fraction of tRNA Ser IGA decreases in rich media, the relative amount of tRNA Ser AGA increases, and UCU codons would be at a relative advantage over UCC codons.…”

“…By comparing β-galactosidase reporters that bear codon swaps of Tyr codon 503 which is required for efficient catalytic activity, in wild-type and strains deleted of the S. pombe tRNA isopentenyltransferase, it was estimated that i 6 A 37 enhances the ability of tRNA Tyr GUA to decode its cognate codon UAC by 3–4 fold [132]. Tyr codons in S. pombe are differentially distributed such that the ratio of UAC-to-UAU is nearly 4.3-to-1 in abundant mRNAs that encode carbon metabolizing energy enzymes but is 0.54-to-1 in low abundance mRNAs, a nearly eight-fold enrichment of the codon with C in the third position in the highly-expressed mRNAs [102]. It was shown that absence of i 6 A 37 specifically on cytosolic tRNA Tyr GUA leads to the carbon source-specific growth deficiency phenotype of fission yeast lacking the tRNA isopenyltransferase, Tit1 [102].…”

“…Tyr codons in S. pombe are differentially distributed such that the ratio of UAC-to-UAU is nearly 4.3-to-1 in abundant mRNAs that encode carbon metabolizing energy enzymes but is 0.54-to-1 in low abundance mRNAs, a nearly eight-fold enrichment of the codon with C in the third position in the highly-expressed mRNAs [102]. It was shown that absence of i 6 A 37 specifically on cytosolic tRNA Tyr GUA leads to the carbon source-specific growth deficiency phenotype of fission yeast lacking the tRNA isopenyltransferase, Tit1 [102]. Overexpression of cytosolic tRNA Tyr GUA in tit1- deletion cells rescues the carbon-specific growth deficiency [102] (schematized in Figure 5B).…”

“…It was shown that absence of i 6 A 37 specifically on cytosolic tRNA Tyr GUA leads to the carbon source-specific growth deficiency phenotype of fission yeast lacking the tRNA isopenyltransferase, Tit1 [102]. Overexpression of cytosolic tRNA Tyr GUA in tit1- deletion cells rescues the carbon-specific growth deficiency [102] (schematized in Figure 5B).…”