Lack of tRNA-i6A modification causes mitochondrial-like metabolic deficiency in S. pombe by limiting activity of cytosolic tRNATyr, 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. Schizosaccharomyces pombe IPTase modifies three different cy-tRNAs(Ser) as well as cy-tRNA(Tyr), cy-tRNA(Trp), and mt-tRNA(Trp). 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 hypomodification-associated 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.

Keywords: mitochondria.

FIGURE 1.

Glycerol and rapamycin slow growth phenotypes of yNB5 (tit1-Δ) are not due to i6A37 hypomodification of mt-tRNA^Trp. (A) Growth of the S. pombe cells indicated to the left (see text) were monitored by spotting serial dilutions onto Edinburgh minimal media (EMM) plates with glucose, or glycerol as the carbon source, or with glucose and containing rapamycin (50 ng/mL). (B–J) RNA analysis including i6A37 modification status of various tRNAs from cells indicated above the lanes using the PHA6 Northern blot assay; (B) EtBr-stained gel showing RNA that was transferred onto membrane and sequentially probed with the ACL and TSL probes indicated to the right of each panel (see text). (K) Quantification of i6A37 modification efficiency of mt-tRNA^Trp from the data from triplicate experiments as calculated using the formula; % modification = [1 − (ACLtit1⁺/BPtit1⁺)/(ACLtit1-Δ/BPtit1-Δ)] × 100 (see Materials and Methods). Error bars reflect triplicate experiments. (L) Quantitation of ATP in yYH1 and yNB5 cells with and without prior growth in a low pharmacological dose of CCCP (20 μM, see text). Equal amounts of protein from each cell lysate were used for ATP colorimetric assay and absorbance was measured at 570 nm.

FIGURE 2.

Ectopic genes encoding cy-tRNAs rescue the metabolic phenotypes of tit1-Δ cells. (A–C) Growth efficiency of various S. pombe cells transformed with empty vector (ev), Tit1⁺, or the genes for tRNAs as indicated to the left (see text) were monitored. This was done by spotting serial dilutions onto EMM plates with glucose (A), or with glucose and containing rapamycin at 50 ng/mL (B), or glycerol as the carbon source (C). +5 tRNAs = one gene each for tRNA^SerAGA, tRNA^SerUGA, tRNA^SerCGA, tRNA^Tyr, and tRNA^Trp on the same plasmid; +3 tRNAs Ser = one each for tRNA^SerAGA, tRNA^SerUGA, tRNA^SerCGA; +2X-Ser-AGA = two genes for tRNA^SerAGA. (D) The cells were also spotted onto EMM with limiting amount of adenine (10 mg/L) to reveal suppressor-tRNA-mediated opal suppression of ade6-704 in the same cells (see text).

FIGURE 3.

Threefold overexpression of tRNA by ectopic tRNA genes. Monitoring expression levels of various tRNAs by Northern hybridization. Annotation above the lanes: (W) yYH1 (wild-type); (M) yNB5 (tit1-Δ); (ev) empty vector; (5tRNAs) tRNA^SerAGA, tRNA^SerUGA, tRNA^SerCGA, tRNA^Tyr, and tRNA^Trp on one plasmid; (2X-Ser-AGA) two consecutive tRNA^SerAGA genes. (A) EtBr-stained gel showing RNA that was transferred onto membrane and sequentially probed for the tRNAs indicated to the right of each panel. (B–F) Four different tRNAs and U5 snRNA were examined using a probe specific for each as indicated to the right. Numbers under panels B–E show quantification, in each case relative to U5, which served as internal loading control.

FIGURE 4.

Evidence of differential mRNA-specific translation efficiency in tit1-Δ cells. (A–D) Two Northern blots of polysome profile fractions, one each from tit1⁺ and tit1-Δ cells, were sequentially probed for the mRNAs indicated to the right. The profiles for skp1 were previously reported (Lamichhane et al. 2011) and are used here as a control (see text).

FIGURE 5.

Different i6A37-cognate codons are uniquely different among mRNA classes of different copy numbers in S. pombe. The top 160 mRNAs sorted according to load enrichment for a particular codon or combination of codons (corrected for mRNA abundance) were plotted as a function of mRNA copy number indicated on the x-axis; number of genes is indicated on the y-axis. ModSer (green bars) represents all codons decoded by i6A37-modified tRNAs^Ser (NGA codons), and the tit1 codon enrichment (red bars) represents all codons decoded by all i6A37-modified tRNAs. The other single codons are also color coded as indicated (see text).

FIGURE 6.

Absence of 2-methylthio modifications in yeast. Total RNAs purified from mouse liver, S. pombe, and S. cerevisiae were subjected to mass spectrometry analysis to detect ms²i⁶A (m/z 382) and i⁶A (m/z 336) modifications. Peaks represent the chromatograms of ms²i⁶A and i⁶A, respectively. Note that ms²i⁶A was not present in S. pombe or S. cerevisiae.