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. 2013 Aug;33(15):2918-29.
doi: 10.1128/MCB.00278-13. Epub 2013 May 28.

Lack of tRNA modification isopentenyl-A37 alters mRNA decoding and causes metabolic deficiencies in fission yeast

Tek N Lamichhane  1 , Nathan H Blewett, Amanda K Crawford, Vera A Cherkasova, James R Iben, Thomas J Begley, Philip J Farabaugh, Richard J Maraia
Affiliations

Lack of tRNA modification isopentenyl-A37 alters mRNA decoding and causes metabolic deficiencies in fission yeast

Tek N Lamichhane et al. Mol Cell Biol. 2013 Aug.

Erratum in

Abstract

tRNA isopentenyltransferases (Tit1) modify tRNA position 37, adjacent to the anticodon, to N6-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-Δ cells by using a β-galactosidase codon-swap reporter whose catalytic activity is sensitive to accurate decoding of codon 503. i6A37 increased the activity of tRNACys at a cognate codon and that of tRNATyr 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-Δ cells. Because subsets of i6A37-tRNAs differ among species, as do their cognate codon-sensitive mRNAs, these genomic variables may underlie associated phenotypic differences.

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Figures

Fig 1
Fig 1
i6A37 increases competitive decoding activity of tRNATyrGUA at a near-cognate codon. (A) Schematic of experimental design. (B) lacZ activities of extracts from yYH1 (tit1+) and yNB5 (tit1-Δ) cells after transformation with lacZ-Y503C. Error bars reflect the ranges for triplicate experiments. (C) Immunoblot detection of FLAG-tagged LacZ-Y503C protein in extracts used for panel B, using anti-FLAG antibody (upper panel). (Lower panel) Ponceau S (Pon S)-stained gel prior to transfer, as a loading control. (D to G) RNA analysis. (D) Ethidium bromide-stained denaturing polyacrylamide gel used to make a blot that was sequentially probed as indicated to the right of panels E to G (see the text). Total RNAs from yYH1 (tit1+) and yNB5 (tit1-Δ) cells were loaded at two concentrations, i.e., 1× (5 μg) and 2× (10 μg), as indicated above the lanes.
Fig 2
Fig 2
i6A37 increases tRNACysGCA activity at its cognate codon. (A) lacZ-Y503C activities as shown in Fig. 1, after transformation of yYH1 (tit1+) and yTL1 (tit1-Δ) cells with empty vector (ev) or vector containing the S. pombe tRNACysGCA-G37 or tRNACysGCA-A37 gene, as indicated below the bars. (B) Immunoblot detection of FLAG-tagged LacZ-Y503C protein in extracts used for panel A, using anti-FLAG antibody (upper panel). (Lower panel) Ponceau S-stained gel prior to transfer, as a loading control. (C to E) Analysis of tRNACysGCA-G37 and tRNACysGCA-A37 levels. (C) Ethidium bromide (EtBr)-stained gel used to make a blot that was sequentially probed as indicated to the right of panels D and E for comparison of expression of ectopic tRNACysGCA-G37 and tRNACysGCA-A37 by use of a body probe that has perfect complementarity to both, with U5 RNA as a loading control. Quantitation of U5 and tRNA levels was done by phosphorimager analysis, and the relative ratios are indicated below the lanes. (F to I) Analysis of i6A37 modification of tRNACysGCA-A37. (F) Ethidium bromide-stained gel. (G) Blot probed for i6A37 modification of tRNACysGCA-A37 by use of the PHA6 assay. See the text for interpretation of the quantification shown under the lanes. (H) Blot probed for tRNACysGCA-G37 and tRNACysGCA-A37 by use of a body probe with perfect complementarity to both. (I) Blot probed for U5 snRNA. Quantitation of U5 and tRNA levels in panels H and I was done by phosphorimager analysis, and the relative ratios are indicated below the lanes. (J to O) ACL and body probing for tRNATrp, tRNATyr, and tRNASerAGA on the same blot, as indicated to the right of the panels.
Fig 3
Fig 3
Deletion of tit1+ causes growth and morphology phenotypes in rapamycin. (A) Comparative serial dilutions of tit1+ and tit1-Δ cells in EMM lacking uracil (left panel; no rapamycin) or EMM lacking uracil but containing 50 μg/ml rapamycin (right panel; + rapamycin), after transformation with empty vector (+ev) or the indicated ectopic plasmid-borne genes: S. pombe tit1+, the tit1-T12A catalytic mutant, and S. cerevisiae MOD5 (see the text). (B) Coomassie blue-stained SDS gel of extracts after labeling with [35S]methionine in the presence or absence of rapamycin (rap) as indicated above the lanes (each extract was loaded in triplicate, at 1×, 2×, and 4× concentrations). (C) The same gel as in panel B, but after drying and exposure to a phosphorimager to visualize 35S-labeled proteins. The asterisks indicate bands specifically absent for rapamycin-treated tit1-Δ cells (see the text). Quantitation results from 3 independent experiments are reported under the lanes. Lanes from gels after Coomassie blue staining were quantified with VisionWorks software, which confirmed that the signals were in the linear range. 35S was quantified with a FLA3000 phosphorimager. The 35S/blue ratios relative to that of tit1+ cells without rapamycin (100%) are reported. (D) Microscopic analysis of tit1+ and tit1-Δ cells containing empty vector (+ev) or ectopic tit1+, as indicated, in the absence (upper panels) or presence (lower panels) of rapamycin. (E) Quantitative analysis of cell length reduction by rapamycin for the three samples in panel D. Lengths of 50 cells from each of the samples represented by the six panels were measured. Cell length in the presence of rapamycin was expressed as % length in the absence of rapamycin. Error bars reflect standard deviations.
Fig 4
Fig 4
Deletion of tit1+ causes i6A37 hypomodification of mt-tRNATrp and growth deficiency in glycerol. (A) Comparative serial dilutions of tit1+ and tit1-Δ cells in EMM lacking uracil (left panel; glucose) or EMM lacking uracil but containing glycerol instead of glucose (right panel; glycerol), after transformation with empty vector (+ev) or the indicated ectopic plasmid-borne genes: S. pombe tit1+, the tit1-T12A catalytic mutant, and S. cerevisiae MOD5. (B) Ethidium bromide-stained gel used to make a blot that was probed as shown in panels C to H. PHA6 Northern blot assays were used to compare the hybridization signals of ACL versus body probes for mitochondrial tRNAs mt-tRNATrp, mt-tRNACys, and mt-tRNASer, as indicated to the right of the panels. (I) Quantitative analysis of i6A37 modification of the 3 mt-tRNAs examined in panels B to H. Error bars reflect standard deviations for 5 experiments. Calculations for each mt-tRNA were performed as follows: % i6A37 modification = [1 − (ACLtit1+/BPtit1+)/(ACLtit1-Δ/BPtit1-Δ)] × 100. ACL, anticodon loop probe; BP, body probe.
Fig 5
Fig 5
Deletion of tit1 leads to decreased translation efficiency. (A) Sucrose gradient sedimentation polysome profiles of extracts from wild-type (black tracing; yYH1 tit1+) and tit1-Δ (red tracing; yNB5) cells. Results of triplicate polysome profile quantitations are shown in the inset; the numbers report the means of three ratios of 60S plus 80S monosomes to polysomes. (B) Ethidium bromide-stained agarose gels of polysome fractions from yYH1 and yNB5 used for Northern blotting. Single and double asterisks to the right indicate 26S and 18S rRNAs, respectively, and “t-” to the left indicates the positions of the tRNAs. (C to F) Northern blots of RNAs isolated from the fractions in panel A, probed for rps1401, rpp103, arp3, and skp1 mRNAs, respectively, as indicated to the right. (G to J) Quantification of the mRNAs in the blots shown in panels C to F. After quantification of individual bands, the relative amounts in the pooled fractions indicated on the x axis were plotted.
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References

    1. Phizicky EM, Hopper AK. 2010. tRNA biology charges to the front. Genes Dev. 24: 1832– 1860 - PMC - PubMed
    1. Bjork GR. 1995. Biosynthesis and function of modified nucleosides, p 165–206 In Söll D, RajBhandary UL. (ed), tRNA: structure, biosynthesis, and function. ASM Press, Washington, DC
    1. Yokoyama S, Nishimura S. 1995. Modified nucleosides and codon recognition, p 207–223 In Söll D, RajBhandary UL. (ed), tRNA: structure, biosynthesis, and function. ASM Press, Washington, DC
    1. Agris PF. 2008. Bringing order to translation: the contributions of transfer RNA anticodon-domain modifications. EMBO Rep. 9: 629– 635 - PMC - PubMed
    1. Agris PF, Vendeix FA, Graham WD. 2007. tRNA's wobble decoding of the genome: 40 years of modification. J. Mol. Biol. 366: 1– 13 - PubMed

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