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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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The Ta0931 gene product (Trm56) can methylate both the elongator tRNAMet transcript and its precursor with an intron. (A) An aliquot of 4 µg of the Ta0931 gene product was analyzed by 15% SDS-PAGE. The gel was stained with Coomassie Brilliant Blue; (B) The Ta0931 gene product and tRNALeuUAG transcript were incubated at 50 °C for 1 h, and then the 14C-methylated nucleotide was analyzed by 2D-TLC; (C) The incorporation of methyl groups into the mature transcript and the precursor of elongator tRNAMet were investigated. The samples were as follows: lane 1, mature transcript of elongator tRNAMet; lane 2, precursor of elongator tRNAMet. The gel was stained with methylene blue (left panel) and the autoradiogram of the same gel was taken (right panel); (D) The incorporation of methyl groups into the mature transcript and the precursor of elongator tRNAMet were measured by the filter assay. Closed and open circles show the incorporation of 14C-methyl groups into the mature transcript and precursor of elongator tRNAMet, respectively.
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ijms-16-00091-f008: The Ta0931 gene product (Trm56) can methylate both the elongator tRNAMet transcript and its precursor with an intron. (A) An aliquot of 4 µg of the Ta0931 gene product was analyzed by 15% SDS-PAGE. The gel was stained with Coomassie Brilliant Blue; (B) The Ta0931 gene product and tRNALeuUAG transcript were incubated at 50 °C for 1 h, and then the 14C-methylated nucleotide was analyzed by 2D-TLC; (C) The incorporation of methyl groups into the mature transcript and the precursor of elongator tRNAMet were investigated. The samples were as follows: lane 1, mature transcript of elongator tRNAMet; lane 2, precursor of elongator tRNAMet. The gel was stained with methylene blue (left panel) and the autoradiogram of the same gel was taken (right panel); (D) The incorporation of methyl groups into the mature transcript and the precursor of elongator tRNAMet were measured by the filter assay. Closed and open circles show the incorporation of 14C-methyl groups into the mature transcript and precursor of elongator tRNAMet, respectively.

Mentions: To analyze whether the Ta0931 gene product was Trm56, the recombinant protein was purified as shown in Figure 8A. The purified Ta0931 methylated the tRNALeuUAG transcript (data not shown) and the methylated nucleotide was identified as pCm (Figure 8B). The Cm modification is only found at position 56 in native tRNALeuUAG (Figure 1C). These results showed that the Ta0931 gene product is Trm56. Neither the S-30 nor the S-100 fraction contained activity that was responsible for introducing the Cm modification into the tRNALeuUAG transcript (Figure 2); however, the genome does encode Trm56. This discrepancy is addressed in the Discussion section. Finally, we investigated the influence of the presence of intron on Trm56 activity. Trm56 methylated both the elongator tRNAMet transcript and its precursor (Figure 8C). However, the methyl group acceptance activity of the precursor was considerably lower than that of the mature transcript (Figure 8D). It should be mentioned that the incubation in Figure 8C was performed for 12 h to show the methylation of the precursor tRNA. These results suggest that the methylation by Trm56 occurs mainly after the removal of the intron. Although the mechanism by which Trm56 recognizes tRNA has not been reported thus far, the results of the current study suggest two possibilities. The first is that the presence of the intron results in steric hindrance that prevents Trm56 binding to the substrate tRNA. The second is that Trm56 directly recognizes the anticodon loop in the tRNA. To clarify the mechanism, further study is required.


Transfer RNA methyltransferases from Thermoplasma acidophilum, a thermoacidophilic archaeon.

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

The Ta0931 gene product (Trm56) can methylate both the elongator tRNAMet transcript and its precursor with an intron. (A) An aliquot of 4 µg of the Ta0931 gene product was analyzed by 15% SDS-PAGE. The gel was stained with Coomassie Brilliant Blue; (B) The Ta0931 gene product and tRNALeuUAG transcript were incubated at 50 °C for 1 h, and then the 14C-methylated nucleotide was analyzed by 2D-TLC; (C) The incorporation of methyl groups into the mature transcript and the precursor of elongator tRNAMet were investigated. The samples were as follows: lane 1, mature transcript of elongator tRNAMet; lane 2, precursor of elongator tRNAMet. The gel was stained with methylene blue (left panel) and the autoradiogram of the same gel was taken (right panel); (D) The incorporation of methyl groups into the mature transcript and the precursor of elongator tRNAMet were measured by the filter assay. Closed and open circles show the incorporation of 14C-methyl groups into the mature transcript and precursor of elongator tRNAMet, respectively.
© Copyright Policy
Related In: Results  -  Collection

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

ijms-16-00091-f008: The Ta0931 gene product (Trm56) can methylate both the elongator tRNAMet transcript and its precursor with an intron. (A) An aliquot of 4 µg of the Ta0931 gene product was analyzed by 15% SDS-PAGE. The gel was stained with Coomassie Brilliant Blue; (B) The Ta0931 gene product and tRNALeuUAG transcript were incubated at 50 °C for 1 h, and then the 14C-methylated nucleotide was analyzed by 2D-TLC; (C) The incorporation of methyl groups into the mature transcript and the precursor of elongator tRNAMet were investigated. The samples were as follows: lane 1, mature transcript of elongator tRNAMet; lane 2, precursor of elongator tRNAMet. The gel was stained with methylene blue (left panel) and the autoradiogram of the same gel was taken (right panel); (D) The incorporation of methyl groups into the mature transcript and the precursor of elongator tRNAMet were measured by the filter assay. Closed and open circles show the incorporation of 14C-methyl groups into the mature transcript and precursor of elongator tRNAMet, respectively.
Mentions: To analyze whether the Ta0931 gene product was Trm56, the recombinant protein was purified as shown in Figure 8A. The purified Ta0931 methylated the tRNALeuUAG transcript (data not shown) and the methylated nucleotide was identified as pCm (Figure 8B). The Cm modification is only found at position 56 in native tRNALeuUAG (Figure 1C). These results showed that the Ta0931 gene product is Trm56. Neither the S-30 nor the S-100 fraction contained activity that was responsible for introducing the Cm modification into the tRNALeuUAG transcript (Figure 2); however, the genome does encode Trm56. This discrepancy is addressed in the Discussion section. Finally, we investigated the influence of the presence of intron on Trm56 activity. Trm56 methylated both the elongator tRNAMet transcript and its precursor (Figure 8C). However, the methyl group acceptance activity of the precursor was considerably lower than that of the mature transcript (Figure 8D). It should be mentioned that the incubation in Figure 8C was performed for 12 h to show the methylation of the precursor tRNA. These results suggest that the methylation by Trm56 occurs mainly after the removal of the intron. Although the mechanism by which Trm56 recognizes tRNA has not been reported thus far, the results of the current study suggest two possibilities. The first is that the presence of the intron results in steric hindrance that prevents Trm56 binding to the substrate tRNA. The second is that Trm56 directly recognizes the anticodon loop in the tRNA. To clarify the mechanism, further study is required.

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