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. 2019 Oct 4;201(21):e00448-19.
doi: 10.1128/JB.00448-19. Print 2019 Nov 1.

Distinct Modified Nucleosides in tRNATrp from the Hyperthermophilic Archaeon Thermococcus kodakarensis and Requirement of tRNA m2G10/m22G10 Methyltransferase (Archaeal Trm11) for Survival at High Temperatures

Akira Hirata  1 , Takeo Suzuki  2 , Tomoko Nagano  1 , Daishiro Fujii  1 , Mizuki Okamoto  1 , Manaka Sora  1 , Todd M Lowe  3 , Tamotsu Kanai  4 , Haruyuki Atomi  4 , Tsutomu Suzuki  5 , Hiroyuki Hori  6
Affiliations

Distinct Modified Nucleosides in tRNATrp from the Hyperthermophilic Archaeon Thermococcus kodakarensis and Requirement of tRNA m2G10/m22G10 Methyltransferase (Archaeal Trm11) for Survival at High Temperatures

Akira Hirata et al. J Bacteriol. .

Abstract

tRNA m2G10/m22G10 methyltransferase (archaeal Trm11) methylates the 2-amino group in guanosine at position 10 in tRNA and forms N2,N2-dimethylguanosine (m22G10) via N2-methylguanosine (m2G10). We determined the complete sequence of tRNATrp, one of the substrate tRNAs for archaeal Trm11 from Thermococcus kodakarensis, a hyperthermophilic archaeon. Liquid chromatography/mass spectrometry following enzymatic digestion of tRNATrp identified 15 types of modified nucleoside at 21 positions. Several modifications were found at novel positions in tRNA, including 2'-O-methylcytidine at position 6, 2-thiocytidine at position 17, 2'-O-methyluridine at position 20, 5,2'-O-dimethylcytidine at position 32, and 2'-O-methylguanosine at position 42. Furthermore, methylwyosine was found at position 37 in this tRNATrp, although 1-methylguanosine is generally found at this location in tRNATrp from other archaea. We constructed trm11 (Δtrm11) and some gene disruptant strains and compared their tRNATrp with that of the wild-type strain, which confirmed the absence of m22G10 and other corresponding modifications, respectively. The lack of 2-methylguanosine (m2G) at position 67 in the trm11 trm14 double disruptant strain suggested that this methylation is mediated by Trm14, which was previously identified as an m2G6 methyltransferase. The Δtrm11 strain grew poorly at 95°C, indicating that archaeal Trm11 is required for T. kodakarensis survival at high temperatures. The m22G10 modification might have effects on stabilization of tRNA and/or correct folding of tRNA at the high temperatures. Collectively, these results provide new clues to the function of modifications and the substrate specificities of modification enzymes in archaeal tRNA, enabling us to propose a strategy for tRNA stabilization of this archaeon at high temperatures.IMPORTANCEThermococcus kodakarensis is a hyperthermophilic archaeon that can grow at 60 to 100°C. The sequence of tRNATrp from this archaeon was determined by liquid chromatography/mass spectrometry. Fifteen types of modified nucleoside were observed at 21 positions, including 5 modifications at novel positions; in addition, methylwyosine at position 37 was newly observed in an archaeal tRNATrp The construction of trm11 (Δtrm11) and other gene disruptant strains confirmed the enzymes responsible for modifications in this tRNA. The lack of 2-methylguanosine (m2G) at position 67 in the trm11 trm14 double disruptant strain suggested that this position is methylated by Trm14, which was previously identified as an m2G6 methyltransferase. The Δtrm11 strain grew poorly at 95°C, indicating that archaeal Trm11 is required for T. kodakarensis survival at high temperatures.

Keywords: archaea; gene disruption; mass spectrometry; tRNA methyltransferase; tRNA modification.

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Figures

FIG 1
FIG 1
Cloverleaf structure of tRNATrp from T. kodakarensis. The modified nucleosides are defined in Table 1.
FIG 2
FIG 2
Position 32 is modified to m5Cm in T. kodakarensis tRNATrp. Top, extracted ion chromatography (XIC) showing coelution of the nucleoside modified at position 32 in tRNATrp from T. kodakaraensis and m5Cm in human cytoplasmic tRNALeuCAA from ALKBH1 knockout cells. Bottom, CID spectrum of m5Cm. The cleavage position of the base-related ion is indicated on the chemical structures.
FIG 3
FIG 3
The trm11 gene disruptant strain shows defective growth at high temperature. (A) Western blot confirming the expression of Trm11 protein in the complemented (Δtrm11 + trm11) strain. Left, proteins in the cell extracts from wild-type, Δtrm11, and complemented strains were separated by 12.5% SDS-PAGE. The gel was stained with Coomassie brilliant blue. Right, proteins were transferred to a membrane, and Western blotting was performed. (B) Growth of the wild-type, Δtrm11, and complemented (Δtrm11 + trm11) strains was measured at 85, 90, 93, and 95°C. Error bars indicate the standard deviations of results of three independent culture experiments.
FIG 4
FIG 4
The thiI and rumA genes are responsible for the formation of s4U8 and 5-methylation of U54, respectively, in tRNATrp. (A) XICs of RNase A-digested fragments containing s4U (top) or U (bottom) at position 8 (arrowheads) are shown. The sequences, m/z, and charge states are indicated on the right. n.d., not detected. (B) XICs of an RNase A-digested fragment containing m5s2U (top) or s2U (bottom) at position 54 (arrowheads). The sequence, m/z, values, and charge states are indicated on the right. (C) CID spectrum of the RNase A-derived fragment from the ΔrumA strain. The sequence and assigned signals are shown in the inset (precursor, doubly charged ions of m/z 1,012.1).
FIG 5
FIG 5
Methylwyosine is present at position 37 in tRNATrp. (A) Predicted biosynthetic pathway of wyosine derivatives in T. kodakarensis. This figure is based on data from a report by de Crécy-Lagard et al. (34). The abbreviations of modified nucleotides are listed in Table 1. The predicted enzymes are indicated. (B) Nucleoside analysis of tRNATrp from wild-type and ΔTYW1 strains. mimG is not observed in the ΔTYW1 sample. (C) In the RNase A fragment from the ΔTYW1 strain, m1G is observed at position 37 instead of mimG37. Asterisks show other eluates with almost the same m/z values. n.d., not detected.
FIG 6
FIG 6
The trm14 gene is responsible for m2G67 formation. (A) Nucleoside analysis of tRNATrp from wild-type (WT) and Δtrm11 Δtrm14 (double disruptant) strains. m22G and m2G are absent in the double disruptant strain. (B) XICs tracing an RNase A-digested fragment containing m2G at position 67 (arrowhead). The sequences, m/z, and charge states are indicated on the right. Asterisks show GmCp (Table 4) with the same m/z value as the m2GCp fragment. n.d.; not detected.
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References

    1. Boccaletto P, Machnicka MA, Purta E, Piątkowski P, Bagiński B, Wirecki TK, de Crécy-Lagard V, Ross R, Limbach PA, Kotter A, Helm M, Bujnicki JM. 2018. MODOMICS: a database of RNA modification pathways. 2017 update. Nucleic Acids Res 46:D303–D307. doi:10.1093/nar/gkx1030. - DOI - PMC - PubMed
    1. Edmonds CG, Crain PF, Gupta R, Hashizume T, Hocart CH, Kowalak JA, Pomerantz SC, Stetter KO, McCloskey JA. 1991. Posttranscriptional modification of tRNA in thermophilic archaea (archaebacteria). J Bacteriol 173:3138–3148. doi:10.1128/jb.173.10.3138-3148.1991. - DOI - PMC - PubMed
    1. McCloskey JA, Graham DE, Zou S, Crain PF, Ibba M, Konisky J, Söll D, Olsen GJ. 2001. Post-transcriptional modification in archaeal tRNAs: identities and phylogenetic relations of nucleotides from mesophilic and hyperthermophilic Methanococcales. Nucleic Acids Res 29:4299–4706. doi:10.1093/nar/29.22.4699. - DOI - PMC - PubMed
    1. Noon KR, Guymon R, Crain PF, McCloskey JA, Thomm M, Lim J, Cavicchioli R. 2003. Influence of temperature on tRNA modification in archaea: Methanococcoides burtonii (optimum growth temperature [Topt], 23 degrees C) and Stetteria hydrogenophila (Topt, 95 degrees C). J Bacteriol 185:5483–5490. doi:10.1128/jb.185.18.5483-5490.2003. - DOI - PMC - PubMed
    1. Sauerwald A, Sitaramaiah D, McCloskey JA, Söll D, Crain PF. 2005. N6-Acetyladenosine: a new modified nucleoside from Methanopyrus kandleri tRNA. FEBS Lett 579:2807–2810. doi:10.1016/j.febslet.2005.04.014. - DOI - PubMed

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