Lack of tRNA Modification Isopentenyl-A37 Alters mRNA Decoding and Causes Metabolic Deficiencies in Fission Yeast

Abstract

d tRNA isopentenyltransferases (Tit1) modify tRNA position 37, adjacent to the anticodon, to N 6 -isopentenyladenosine (i6A37) in all cells, yet the tRNA subsets selected for modification vary among species, and their relevance to phenotypes is unknown. We examined the function of i6A37 in Schizosaccharomyces pombe tit1؉ and tit1-⌬ cells by using a ␤-galactosidase codon-swap reporter whose catalytic activity is sensitive to accurate decoding of codon 503. i6A37 increased the activity of tRNA Cys at a cognate codon and that of tRNA Tyr at a near-cognate codon, suggesting that i6A37 promotes decoding activity generally and increases fidelity at cognate codons while decreasing fidelity at noncognate codons. S. pombe cells lacking tit1 ؉ exhibit slow growth in glycerol or rapamycin. While existing data link wobble base U34 modifications to translation of functionally related mRNAs, whether this might extend to the anticodon-adjacent position 37 was unknown. Indeed, we found a biased presence of i6A37-cognate codons in high-abundance mRNAs for ribosome subunits and energy metabolism, congruent with the observed phenotypes and the idea that i6A37 promotes translational efficiency. Polysome profiles confirmed the decreased translational efficiency of mRNAs in tit1-⌬ cells. Because subsets of i6A37-tRNAs differ among species, as do their cognate codon-sensitive mRNAs, these genomic variables may underlie associated phenotypic differences.

Protein folding and tRNA biology

“…The PHA6 northern blot assay is based on a DNA probe complementary to the anticodon loop which hybridizes better in the absence of i6A37 and a probe complementary to a different region of the same tRNA as a quantitative internal control. The procedure can be used to calibrate and measure differences in i6A37 modification (Lamichhane et al 2011(Lamichhane et al , 2013a(Lamichhane et al , b, 2016Yarham et al 2014).…”

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

“…Deletion of the IPTase decreases sup-tRNA activity in fission (tRNA Ser ) and budding yeast (tRNA Tyr ) causing loss of TMS phenotype (Laten et al 1978;Janner et al 1980;Laten 1984;Dihanich et al 1987; see Kohli et al 1989) with no effect on the levels of the sup-tRNA (Lamichhane et al 2013a). Another phenotype of S. pombe tit1-Δ is sensitivity to rapamycin, an inhibitor of TOR kinase (Lamichhane et al 2013a).…”

“…Schizosaccharomyces pombe has five i6A37-cy-tRNAs, comprised of three cy- (Chan and Lowe 2009;Lamichhane et al 2011). An assay in S. pombe dependent on decoding of a Tyr codon in the active center of β-galactosidase revealed that loss of i6A37 decreased cy-tRNA Tyr specific activity by approximately threefold (Lamichhane et al 2013a). A more facile and widely used assay is tRNA-mediated suppression (TMS) in which the activity of a suppressor-tRNA (suptRNA) to decode/suppress a premature stop codon in a reporter mRNA encoding a metabolic enzyme in an adenine synthetic pathway is monitored by red-white colony color (for review, see Rijal et al 2015).…”

“…A tRNA modification in bacteria, yeast, and animals is isopentenyl-N 6 -A37 (i6A37). In eukaryotes, i6A37 is found on cytosolic and mitochondrial tRNAs (cy-and mt-tRNAs) (Dihanich et al 1987;Lakowski and Hekimi 1996), intriguingly, on different subsets of tRNAs in different species (Lamichhane et al 2011(Lamichhane et al , 2013a for review, see Maraia and Iben 2014). In bacteria, i6A37 is hypermodified to 2-methylthio-N 6 -isopentenyl-A37 (ms2i6A37).…”

“…A more facile and widely used assay is tRNA-mediated suppression (TMS) in which the activity of a suppressor-tRNA (suptRNA) to decode/suppress a premature stop codon in a reporter mRNA encoding a metabolic enzyme in an adenine synthetic pathway is monitored by red-white colony color (for review, see Rijal et al 2015). Deletion of the IPTase decreases sup-tRNA activity in fission (tRNA Ser ) and budding yeast (tRNA Tyr ) causing loss of TMS phenotype (Laten et al 1978;Janner et al 1980;Laten 1984;Dihanich et al 1987; see Kohli et al 1989) with no effect on the levels of the sup-tRNA (Lamichhane et al 2013a). Another phenotype of S. pombe tit1-Δ is sensitivity to rapamycin, an inhibitor of TOR kinase (Lamichhane et al 2013a).…”