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Transfer RNA methyltransferases from Thermoplasma acidophilum, a thermoacidophilic archaeon.

Kawamura T, Anraku R, Hasegawa T, Tomikawa C, Hori H - Int J Mol Sci (2014)

Bottom Line: We found that the Ta0997, Ta0931, and Ta0836 genes of T. acidophilum encode Trm1, Trm56 and Trm5, respectively.However, the speed of methyl-transfer by Trm56 to the precursor RNA was considerably slower than that to the mature transcript, which suggests that Trm56 acts mainly on the transcript after the intron has been removed.Moreover, cellular arrangements of the tRNA methyltransferases in T. acidophilum are discussed.

View Article: PubMed Central - PubMed

Affiliation: Department of Materials Science and Biotechnology, Graduate School of Science and Engineering, Ehime Univsersity, Bunkyo 3, Matsuyama, Ehime 790-8577, Japan. t.kwmr.0115@gmail.com.

ABSTRACT
We investigated tRNA methyltransferase activities in crude cell extracts from the thermoacidophilic archaeon Thermoplasma acidophilum. We analyzed the modified nucleosides in native initiator and elongator tRNAMet, predicted the candidate genes for the tRNA methyltransferases on the basis of the tRNAMet and tRNALeu sequences, and characterized Trm5, Trm1 and Trm56 by purifying recombinant proteins. We found that the Ta0997, Ta0931, and Ta0836 genes of T. acidophilum encode Trm1, Trm56 and Trm5, respectively. Initiator tRNAMet from T. acidophilum strain HO-62 contained G+, m1I, and m22G, which were not reported previously in this tRNA, and the m2G26 and m22G26 were formed by Trm1. In the case of elongator tRNAMet, our analysis showed that the previously unidentified G modification at position 26 was a mixture of m2G and m22G, and that they were also generated by Trm1. Furthermore, purified Trm1 and Trm56 could methylate the precursor of elongator tRNAMet, which has an intron at the canonical position. However, the speed of methyl-transfer by Trm56 to the precursor RNA was considerably slower than that to the mature transcript, which suggests that Trm56 acts mainly on the transcript after the intron has been removed. Moreover, cellular arrangements of the tRNA methyltransferases in T. acidophilum are discussed.

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Related in: MedlinePlus

Activities of tRNA methyltransferases in the crude cell extracts. The activities of tRNA methyltransferases in the S-30 (A) and S-100 (B) fractions were analyzed by 2D-TLC. 14C-methylated nucleotides were monitored by autoradiography. The solvent systems were as follows: first dimension, isobutyric acid, ammonia, water, 66/1/33 v/v/v; second dimension, isopropyl alcohol, HCl, water, 70/15/15 v/v/v.
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ijms-16-00091-f002: Activities of tRNA methyltransferases in the crude cell extracts. The activities of tRNA methyltransferases in the S-30 (A) and S-100 (B) fractions were analyzed by 2D-TLC. 14C-methylated nucleotides were monitored by autoradiography. The solvent systems were as follows: first dimension, isobutyric acid, ammonia, water, 66/1/33 v/v/v; second dimension, isopropyl alcohol, HCl, water, 70/15/15 v/v/v.

Mentions: Next, we tested tRNA methyltransferase activities in crude extract from T. acidophilum cells. The supernatant fraction from centrifugation at 30,000× g (S-30) was prepared and then the tRNALeuUAG transcript was subjected to methylation by the S-30 extract with 14C-S-adenosyl-l-methionine (AdoMet) as the methyl group donor. The methylated tRNA was digested completely with nuclease P1 and then the resultant 14C-methylated nucleotides were analyzed by two-dimensional thin-layer chromatography (2D-TLC). As shown in Figure 2A, four 14C-methylated nucleotides (pm1G, pm2G, pm22G and pm6A) could be detected. On the basis of the sequence of tRNALeuUAG (Figure 1C) and the candidate enzymes (Table 1), pm1G, pm2G and pm22G, and pm6A were expected to be derived from the activities of Trm5, Trm1, and TrmI, respectively: pm6A could be converted from pm1A non-enzymatically [46]. However, unexpectedly, pCm and pm7G were not detected. In general, the formation of pCm by Trm56 is one of the most common tRNA methyltransferase activities found in crude extract from archaeal cells. For example, it was reported that Trm56 activity in relation to the formation of pCm56 is clearly detected in cell extract from Pyrococcus furiosus [47]. Analysis of the T. acidophilum proteome revealed that various proteins form several large (more than 300 kDa) protein complexes and that some protein complexes might interact with the membrane [48]. Consequently, Trm56 and an unknown tRNA (m7G49) methyltransferase might be included in protein complexes and precipitated by centrifugation at 30,000× g. We tested several buffer conditions such as variations in pH, components, detergents, and salt concentrations (data not shown). However, to date, we have not detected enzyme activities responsible for the formation of pCm and pm7G in crude extract from T. acidophilum cells, though there are other possibilities; while we used aluminum oxide to prepare the extract in this experiment, other methods for preparation of cell extract should be tested. In addition, tRNA methyltransferases in the crude extract have different affinities for 14C-AdoMet. The concentration of 14C-AdoMet in the experiment was 19.5 µM. In this case, tRNA methyltransferases, which have relatively high affinity for AdoMet, might preferentially consume the 14C-AdoMet. When the supernatant fraction from centrifugation at 100,000× g (S-100) was used as the cell extract instead of the S-30 fraction, the findings were even more marked: only the formation of pm1G was detectable (Figure 2B). Given that tRNA methyltransferases have a general affinity for RNA, the enzymes often bind to ribosomes and are precipitated by centrifugation at 100,000× g. In fact, the majority of TrmI from Thermus thermophilus [49] is precipitated by centrifugation at 100,000× g [50]. However, our findings with the extract from T. acidophilum are unprecedented. In the current study, we characterized tRNA methyltransferases by analyzing purified recombinant proteins. However, these enzymes might interact with other proteins and form large protein complexes in living cells.


Transfer RNA methyltransferases from Thermoplasma acidophilum, a thermoacidophilic archaeon.

Kawamura T, Anraku R, Hasegawa T, Tomikawa C, Hori H - Int J Mol Sci (2014)

Activities of tRNA methyltransferases in the crude cell extracts. The activities of tRNA methyltransferases in the S-30 (A) and S-100 (B) fractions were analyzed by 2D-TLC. 14C-methylated nucleotides were monitored by autoradiography. The solvent systems were as follows: first dimension, isobutyric acid, ammonia, water, 66/1/33 v/v/v; second dimension, isopropyl alcohol, HCl, water, 70/15/15 v/v/v.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4307237&req=5

ijms-16-00091-f002: Activities of tRNA methyltransferases in the crude cell extracts. The activities of tRNA methyltransferases in the S-30 (A) and S-100 (B) fractions were analyzed by 2D-TLC. 14C-methylated nucleotides were monitored by autoradiography. The solvent systems were as follows: first dimension, isobutyric acid, ammonia, water, 66/1/33 v/v/v; second dimension, isopropyl alcohol, HCl, water, 70/15/15 v/v/v.
Mentions: Next, we tested tRNA methyltransferase activities in crude extract from T. acidophilum cells. The supernatant fraction from centrifugation at 30,000× g (S-30) was prepared and then the tRNALeuUAG transcript was subjected to methylation by the S-30 extract with 14C-S-adenosyl-l-methionine (AdoMet) as the methyl group donor. The methylated tRNA was digested completely with nuclease P1 and then the resultant 14C-methylated nucleotides were analyzed by two-dimensional thin-layer chromatography (2D-TLC). As shown in Figure 2A, four 14C-methylated nucleotides (pm1G, pm2G, pm22G and pm6A) could be detected. On the basis of the sequence of tRNALeuUAG (Figure 1C) and the candidate enzymes (Table 1), pm1G, pm2G and pm22G, and pm6A were expected to be derived from the activities of Trm5, Trm1, and TrmI, respectively: pm6A could be converted from pm1A non-enzymatically [46]. However, unexpectedly, pCm and pm7G were not detected. In general, the formation of pCm by Trm56 is one of the most common tRNA methyltransferase activities found in crude extract from archaeal cells. For example, it was reported that Trm56 activity in relation to the formation of pCm56 is clearly detected in cell extract from Pyrococcus furiosus [47]. Analysis of the T. acidophilum proteome revealed that various proteins form several large (more than 300 kDa) protein complexes and that some protein complexes might interact with the membrane [48]. Consequently, Trm56 and an unknown tRNA (m7G49) methyltransferase might be included in protein complexes and precipitated by centrifugation at 30,000× g. We tested several buffer conditions such as variations in pH, components, detergents, and salt concentrations (data not shown). However, to date, we have not detected enzyme activities responsible for the formation of pCm and pm7G in crude extract from T. acidophilum cells, though there are other possibilities; while we used aluminum oxide to prepare the extract in this experiment, other methods for preparation of cell extract should be tested. In addition, tRNA methyltransferases in the crude extract have different affinities for 14C-AdoMet. The concentration of 14C-AdoMet in the experiment was 19.5 µM. In this case, tRNA methyltransferases, which have relatively high affinity for AdoMet, might preferentially consume the 14C-AdoMet. When the supernatant fraction from centrifugation at 100,000× g (S-100) was used as the cell extract instead of the S-30 fraction, the findings were even more marked: only the formation of pm1G was detectable (Figure 2B). Given that tRNA methyltransferases have a general affinity for RNA, the enzymes often bind to ribosomes and are precipitated by centrifugation at 100,000× g. In fact, the majority of TrmI from Thermus thermophilus [49] is precipitated by centrifugation at 100,000× g [50]. However, our findings with the extract from T. acidophilum are unprecedented. In the current study, we characterized tRNA methyltransferases by analyzing purified recombinant proteins. However, these enzymes might interact with other proteins and form large protein complexes in living cells.

Bottom Line: We found that the Ta0997, Ta0931, and Ta0836 genes of T. acidophilum encode Trm1, Trm56 and Trm5, respectively.However, the speed of methyl-transfer by Trm56 to the precursor RNA was considerably slower than that to the mature transcript, which suggests that Trm56 acts mainly on the transcript after the intron has been removed.Moreover, cellular arrangements of the tRNA methyltransferases in T. acidophilum are discussed.

View Article: PubMed Central - PubMed

Affiliation: Department of Materials Science and Biotechnology, Graduate School of Science and Engineering, Ehime Univsersity, Bunkyo 3, Matsuyama, Ehime 790-8577, Japan. t.kwmr.0115@gmail.com.

ABSTRACT
We investigated tRNA methyltransferase activities in crude cell extracts from the thermoacidophilic archaeon Thermoplasma acidophilum. We analyzed the modified nucleosides in native initiator and elongator tRNAMet, predicted the candidate genes for the tRNA methyltransferases on the basis of the tRNAMet and tRNALeu sequences, and characterized Trm5, Trm1 and Trm56 by purifying recombinant proteins. We found that the Ta0997, Ta0931, and Ta0836 genes of T. acidophilum encode Trm1, Trm56 and Trm5, respectively. Initiator tRNAMet from T. acidophilum strain HO-62 contained G+, m1I, and m22G, which were not reported previously in this tRNA, and the m2G26 and m22G26 were formed by Trm1. In the case of elongator tRNAMet, our analysis showed that the previously unidentified G modification at position 26 was a mixture of m2G and m22G, and that they were also generated by Trm1. Furthermore, purified Trm1 and Trm56 could methylate the precursor of elongator tRNAMet, which has an intron at the canonical position. However, the speed of methyl-transfer by Trm56 to the precursor RNA was considerably slower than that to the mature transcript, which suggests that Trm56 acts mainly on the transcript after the intron has been removed. Moreover, cellular arrangements of the tRNA methyltransferases in T. acidophilum are discussed.

Show MeSH
Related in: MedlinePlus