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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

T. acidophilum Trm1 (the Ta0997 gene product) is a single-site-specific enzyme. (A) The T. acidophilum Ta0997 gene product (left), T. kodakaraensis Trm1 (center) and A. aeolicus Trm1 (right) (2 µg each) were analyzed by 15% SDS-PAGE. The gel was stained with Coomassie Brilliant Blue; (B) The tRNALeuUAG transcript was methylated by the Ta0997 gene product and then the generated methylated nucleotide was analyzed by 2D-TLC; (C) The sequences of S. cerevisiae tRNAPhe, A. aeolicus tRNATyr and A. aeolicus tRNATyr A26G, G27A are depicted by cloverleaf structures. These tRNA transcripts were previously used to determine the site specificity of A. aeolicus Trm1, which is a multi-site-specific Trm1. The nucleotides at position 26 in the tRNA transcripts are colored red. The transcript numbers (1, 2, and 3) correspond to the lane numbers in panels (D–F). (D) The tRNA transcripts (0.1 A260 units each) were incubated with the Ta0997 gene product (T. acidophilum Trm1) and 14C-AdoMet at 50 °C for 5 min, separated by 10% PAGE (7 M urea), and then the gel was analyzed by autoradiography. The left and right panels show the gel stained with methylene blue and its autoradiogram, respectively. T. kodakaraensis (E) and A. aeolicus (F) Trm1 proteins were analyzed by the same method.
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ijms-16-00091-f005: T. acidophilum Trm1 (the Ta0997 gene product) is a single-site-specific enzyme. (A) The T. acidophilum Ta0997 gene product (left), T. kodakaraensis Trm1 (center) and A. aeolicus Trm1 (right) (2 µg each) were analyzed by 15% SDS-PAGE. The gel was stained with Coomassie Brilliant Blue; (B) The tRNALeuUAG transcript was methylated by the Ta0997 gene product and then the generated methylated nucleotide was analyzed by 2D-TLC; (C) The sequences of S. cerevisiae tRNAPhe, A. aeolicus tRNATyr and A. aeolicus tRNATyr A26G, G27A are depicted by cloverleaf structures. These tRNA transcripts were previously used to determine the site specificity of A. aeolicus Trm1, which is a multi-site-specific Trm1. The nucleotides at position 26 in the tRNA transcripts are colored red. The transcript numbers (1, 2, and 3) correspond to the lane numbers in panels (D–F). (D) The tRNA transcripts (0.1 A260 units each) were incubated with the Ta0997 gene product (T. acidophilum Trm1) and 14C-AdoMet at 50 °C for 5 min, separated by 10% PAGE (7 M urea), and then the gel was analyzed by autoradiography. The left and right panels show the gel stained with methylene blue and its autoradiogram, respectively. T. kodakaraensis (E) and A. aeolicus (F) Trm1 proteins were analyzed by the same method.

Mentions: The m22G modification was observed only at position 26 in tRNALeuUAG ([28] and Figure 1C). Trm1 transfers two methyl groups to the 2-amino group in the target guanine and m2G is formed as an intermediate [57,58]. Consequently, the m2G and m22G modifications in tRNALeuUAG transcript by the S-30 (Figure 2A) were expected to be derived from Trm1 activity. Trm1 enzymes can be divided into two types on the basis of their specificity for the target guanosine(s). One is a single-site-specific Trm1, which modifies only G26 and is found in eukaryotes and archaea [57,58,59,60]. The second is a multi-site-specific Trm1, which modifies both G26 and G27 and is found in the hyperthermophilic eubacterium, A. aeolicus [52]. In addition to the Ta0997 gene product (Figure 5A lane 1), we prepared two types of Trm1 enzyme from Thermococcus kodakarensis (Figure 5A lane 2) and A. aeolicus (Figure 5A lane 3) as controls. The Ta0997 gene product methylated the tRNALeuUAG transcript (data not shown) and 14C-nucleotide analysis revealed that the modified nucleotide was pm22G (Figure 5B). These results showed that the Ta0997 gene product is the T. acidophilum Trm1 protein. To distinguish the site specificity, yeast tRNAPhe and A. aeolicus tRNATyr transcripts were prepared (Figure 5C). These tRNA transcripts were used previously to assess the site specificity of A. aeolicus Trm1 [52]. Yeast tRNAPhe contains the sequence G26C27, whereas A. aeolicus tRNATyr contains the sequence A26G27. In addition, the A. aeolicus tRNATyr A26G, G27A mutant transcript has the sequence G26A27. As shown in Figure 5D, T. acidophilum Trm1 methylated yeast tRNAPhe and A. aeolicus tRNATyr A26G, G27A transcripts, which contain G26. In contrast, T. acidophilum Trm1 did not methylate the wild-type tRNATyr transcript (Figure 5D center), which contains A26. Thus, these results demonstrate that T. acidophilum Trm1 is a single-site-specific Trm1, which methylates only G26. Similar to T. acidophilum Trm1, T. kodakarensis Trm1 methylated only the yeast tRNAPhe and A. aeolicus tRNATyr A26G, G27A transcripts (Figure 5E). In contrast, A. aeolicus Trm1 methylated all the transcripts (Figure 5F), which indicates that A. aeolicus Trm1 has multi-site specificity.


Transfer RNA methyltransferases from Thermoplasma acidophilum, a thermoacidophilic archaeon.

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

T. acidophilum Trm1 (the Ta0997 gene product) is a single-site-specific enzyme. (A) The T. acidophilum Ta0997 gene product (left), T. kodakaraensis Trm1 (center) and A. aeolicus Trm1 (right) (2 µg each) were analyzed by 15% SDS-PAGE. The gel was stained with Coomassie Brilliant Blue; (B) The tRNALeuUAG transcript was methylated by the Ta0997 gene product and then the generated methylated nucleotide was analyzed by 2D-TLC; (C) The sequences of S. cerevisiae tRNAPhe, A. aeolicus tRNATyr and A. aeolicus tRNATyr A26G, G27A are depicted by cloverleaf structures. These tRNA transcripts were previously used to determine the site specificity of A. aeolicus Trm1, which is a multi-site-specific Trm1. The nucleotides at position 26 in the tRNA transcripts are colored red. The transcript numbers (1, 2, and 3) correspond to the lane numbers in panels (D–F). (D) The tRNA transcripts (0.1 A260 units each) were incubated with the Ta0997 gene product (T. acidophilum Trm1) and 14C-AdoMet at 50 °C for 5 min, separated by 10% PAGE (7 M urea), and then the gel was analyzed by autoradiography. The left and right panels show the gel stained with methylene blue and its autoradiogram, respectively. T. kodakaraensis (E) and A. aeolicus (F) Trm1 proteins were analyzed by the same method.
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Related In: Results  -  Collection

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Show All Figures
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ijms-16-00091-f005: T. acidophilum Trm1 (the Ta0997 gene product) is a single-site-specific enzyme. (A) The T. acidophilum Ta0997 gene product (left), T. kodakaraensis Trm1 (center) and A. aeolicus Trm1 (right) (2 µg each) were analyzed by 15% SDS-PAGE. The gel was stained with Coomassie Brilliant Blue; (B) The tRNALeuUAG transcript was methylated by the Ta0997 gene product and then the generated methylated nucleotide was analyzed by 2D-TLC; (C) The sequences of S. cerevisiae tRNAPhe, A. aeolicus tRNATyr and A. aeolicus tRNATyr A26G, G27A are depicted by cloverleaf structures. These tRNA transcripts were previously used to determine the site specificity of A. aeolicus Trm1, which is a multi-site-specific Trm1. The nucleotides at position 26 in the tRNA transcripts are colored red. The transcript numbers (1, 2, and 3) correspond to the lane numbers in panels (D–F). (D) The tRNA transcripts (0.1 A260 units each) were incubated with the Ta0997 gene product (T. acidophilum Trm1) and 14C-AdoMet at 50 °C for 5 min, separated by 10% PAGE (7 M urea), and then the gel was analyzed by autoradiography. The left and right panels show the gel stained with methylene blue and its autoradiogram, respectively. T. kodakaraensis (E) and A. aeolicus (F) Trm1 proteins were analyzed by the same method.
Mentions: The m22G modification was observed only at position 26 in tRNALeuUAG ([28] and Figure 1C). Trm1 transfers two methyl groups to the 2-amino group in the target guanine and m2G is formed as an intermediate [57,58]. Consequently, the m2G and m22G modifications in tRNALeuUAG transcript by the S-30 (Figure 2A) were expected to be derived from Trm1 activity. Trm1 enzymes can be divided into two types on the basis of their specificity for the target guanosine(s). One is a single-site-specific Trm1, which modifies only G26 and is found in eukaryotes and archaea [57,58,59,60]. The second is a multi-site-specific Trm1, which modifies both G26 and G27 and is found in the hyperthermophilic eubacterium, A. aeolicus [52]. In addition to the Ta0997 gene product (Figure 5A lane 1), we prepared two types of Trm1 enzyme from Thermococcus kodakarensis (Figure 5A lane 2) and A. aeolicus (Figure 5A lane 3) as controls. The Ta0997 gene product methylated the tRNALeuUAG transcript (data not shown) and 14C-nucleotide analysis revealed that the modified nucleotide was pm22G (Figure 5B). These results showed that the Ta0997 gene product is the T. acidophilum Trm1 protein. To distinguish the site specificity, yeast tRNAPhe and A. aeolicus tRNATyr transcripts were prepared (Figure 5C). These tRNA transcripts were used previously to assess the site specificity of A. aeolicus Trm1 [52]. Yeast tRNAPhe contains the sequence G26C27, whereas A. aeolicus tRNATyr contains the sequence A26G27. In addition, the A. aeolicus tRNATyr A26G, G27A mutant transcript has the sequence G26A27. As shown in Figure 5D, T. acidophilum Trm1 methylated yeast tRNAPhe and A. aeolicus tRNATyr A26G, G27A transcripts, which contain G26. In contrast, T. acidophilum Trm1 did not methylate the wild-type tRNATyr transcript (Figure 5D center), which contains A26. Thus, these results demonstrate that T. acidophilum Trm1 is a single-site-specific Trm1, which methylates only G26. Similar to T. acidophilum Trm1, T. kodakarensis Trm1 methylated only the yeast tRNAPhe and A. aeolicus tRNATyr A26G, G27A transcripts (Figure 5E). In contrast, A. aeolicus Trm1 methylated all the transcripts (Figure 5F), which indicates that A. aeolicus Trm1 has multi-site specificity.

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