Plasticity and diversity of tRNA anticodon determinants of substrate recognition by eukaryotic A37 isopentenyltransferases

Abstract

The N 6 -(isopentenyl)adenosine (i 6 A) modification of some tRNAs at position A37 is found in all kingdoms and facilitates codonspecific mRNA decoding, but occurs in different subsets of tRNAs in different species. Here we examine yeasts' tRNA isopentenyltransferases (i.e., dimethylallyltransferase, DMATase, members of the D 2 -isopentenylpyrophosphate transferase, IPPT superfamily) encoded by tit1 + in Schizosaccharomyces pombe and MOD5 in Saccharomyces cerevisiae, whose homologs are Escherichia coli miaA, the human tumor suppressor TRIT1, and the Caenorhabditis elegans life-span gene product GRO-1. A major determinant of miaA activity is known to be the single-stranded tRNA sequence, A36A37A38, in a stem-loop. tRNA Trp CCA from either yeast is a Tit1p substrate, but neither is a Mod5p substrate despite the presence of A36A37A38. We show that Tit1p accommodates a broader range of substrates than Mod5p. tRNA Trp CCA is distinct from Mod5p substrates, which we sort into two classes based on the presence of G at position 34 and other elements. A single substitution of C34 to G converts tRNA Trp CCA to a Mod5p substrate in vitro and in vivo, consistent with amino acid contacts to G34 in existing Mod5p-tRNA Cys GCA crystal structures. Mutation of Mod5p in its G34 recognition loop region debilitates it differentially for its G34 (class I) substrates. Multiple alignments reveal that the G34 recognition loop sequence of Mod5p differs significantly from Tit1p, which more resembles human TRIT1 and other DMATases. We show that TRIT1 can also modify tRNA Trp CCA consistent with broad recognition similar to Tit1p. This study illustrates previously unappreciated molecular plasticity and biological diversity of the tRNA-isopentenyltransferase system of eukaryotes.

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).…”

The Levels of a Universally Conserved tRNA Modification Regulate Cell Growth

“…Our in silico analysis and the interspecies complementation tests suggest that Drosophila Tcs3 is implicated in t 6 A synthesis. This modification was detected using an adaptation of a hybridization method used to detect the presence of N 6 -isopentenyl-adenosine (i 6 A), another modification found at position 37 of specific tRNAs (43). Briefly, when the threonylcarbamoyl moiety is present in position Ala-37, it impairs Watson-Crick pairing between the tRNA and the specific DNA FIGURE 2.…”

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

“…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).…”

“…Yeast lacking IPTase grow normally in standard media (Laten et al 1978;Janner et al 1980;Laten 1984;Dihanich et al 1987). 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).…”