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Base methylations in the double-stranded RNA by a fused methyltransferase bearing unwinding activity.

Kimura S, Ikeuchi Y, Kitahara K, Sakaguchi Y, Suzuki T, Suzuki T - Nucleic Acids Res. (2011)

Bottom Line: The N-terminal RlmL activity for m(2)G2445 formation was significantly enhanced by the C-terminal RlmK.Moreover, RlmKL had an unwinding activity of Helix 74, facilitating cooperative methylations of m(7)G2069 and m(2)G2445 during biogenesis of 50S subunit.In fact, we observed that RlmKL was involved in the efficient assembly of 50S subunit in a mutant strain lacking an RNA helicase deaD.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry and Biotechnology, Graduate School of Engineering, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8656, Japan.

ABSTRACT
Modifications of rRNAs are clustered in functional regions of the ribosome. In Helix 74 of Escherichia coli 23S rRNA, guanosines at positions 2069 and 2445 are modified to 7-methylguanosine(m(7)G) and N(2)-methylguanosine(m(2)G), respectively. We searched for the gene responsible for m(7)G2069 formation, and identified rlmL, which encodes the methyltransferase for m(2)G2445, as responsible for the biogenesis of m(7)G2069. In vitro methylation of rRNA revealed that rlmL encodes a fused methyltransferase responsible for forming both m(7)G2069 and m(2)G2445. We renamed the gene rlmKL. The N-terminal RlmL activity for m(2)G2445 formation was significantly enhanced by the C-terminal RlmK. Moreover, RlmKL had an unwinding activity of Helix 74, facilitating cooperative methylations of m(7)G2069 and m(2)G2445 during biogenesis of 50S subunit. In fact, we observed that RlmKL was involved in the efficient assembly of 50S subunit in a mutant strain lacking an RNA helicase deaD.

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Complementation study of m7G2069 and m2G2445 formation by plasmid-encoded RlmKL and mutants. (A) Domain structure of E. coli RlmKL. RlmKL consists of two methyltransferase domains, COG0116 and COG1092. We designated the N-terminal domain (COG0116) as RlmL and the C-terminal domain (COG1092) as RlmK. Each domain has an Ado-Met-dependent methyltransferase motif, UPF0020 in the RlmL domain and Cons_hypoth 95 in the RlmK domain. A THUMP domain was found in the N-terminal region of the RlmL domain. (B) Complementation of the ΔrlmKL strain by introducing plasmid-encoded RlmKL or RlmKL mutants. On the left hand side, the domain structures and the mutation positions of RlmKL are shown. On the right hand side, mass chromatograms detecting the 16-mer fragment carrying m7G2069 (m/z 1706) and the hexamer fragment carrying m2G2445 (m/z 1024), respectively, are shown for the ΔrlmKL strain transformed with pRlmKL (top panels), pRlmKL N309A (second panels), pRlmKL N397A (third panels), pRlmK (fourth panels), pRlmKL D568A (fifth panels) and pRlmL (bottom panels). (C) Complementation of the E. coli ΔrlmKL strain by introducing N. meningtidis RlmL (NMB0445) or RlmK (NMB1367). Mass chromatograms on the right panels are the same as in (B).
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gkr1287-F3: Complementation study of m7G2069 and m2G2445 formation by plasmid-encoded RlmKL and mutants. (A) Domain structure of E. coli RlmKL. RlmKL consists of two methyltransferase domains, COG0116 and COG1092. We designated the N-terminal domain (COG0116) as RlmL and the C-terminal domain (COG1092) as RlmK. Each domain has an Ado-Met-dependent methyltransferase motif, UPF0020 in the RlmL domain and Cons_hypoth 95 in the RlmK domain. A THUMP domain was found in the N-terminal region of the RlmL domain. (B) Complementation of the ΔrlmKL strain by introducing plasmid-encoded RlmKL or RlmKL mutants. On the left hand side, the domain structures and the mutation positions of RlmKL are shown. On the right hand side, mass chromatograms detecting the 16-mer fragment carrying m7G2069 (m/z 1706) and the hexamer fragment carrying m2G2445 (m/z 1024), respectively, are shown for the ΔrlmKL strain transformed with pRlmKL (top panels), pRlmKL N309A (second panels), pRlmKL N397A (third panels), pRlmK (fourth panels), pRlmKL D568A (fifth panels) and pRlmL (bottom panels). (C) Complementation of the E. coli ΔrlmKL strain by introducing N. meningtidis RlmL (NMB0445) or RlmK (NMB1367). Mass chromatograms on the right panels are the same as in (B).

Mentions: Escherichia coli RlmKL is a fused methyltransferase consisting of 702 amino acid residues, containing a thiouridine synthases, methylases and PSUSs (THUMP) domain and two methyltransferase domains (Figure 3A). According to the cluster of orthologous group (COG) database (42), the N-terminal domain (NTD) and C-terminal domain (CTD) of RlmKL have been annotated as COG0116 and COG1092, respectively. Each domain has an Ado-Met-dependent methyltransferase motif (Figure 3A). The rlmKL gene is conserved in γ-proteobacteria (Supplementary Figure S1). However, in β-proteobacteria, Neisseria meningitidis bears COG0116 and COG1092 as separate proteins encoded at different genomic loci (Supplementary Figure S1). Since rlmKL appears to be responsible for two different methyl modifications, we hypothesized that each methyltransferase domain catalyzes the formation of one modification, either m7G2069 or m2G2445. To examine this possibility, we carried out complementation tests for the two methyl modifications in the ΔrlmKL strain by introducing various rlmKL mutants. We constructed rlmKL mutants in which conserved amino acid residues were substituted with Ala, including D195A, N309A, N397A, R530A, D568A and D597A. As a positive control, introduction of plasmid-encoded rlmKL into the ΔrlmKL strain restored both methyl modifications (Figure 3B). Subsequently, each of the rlmKL mutants was introduced into the ΔrlmKL strain, and formation of the methyl modifications was examined. The N309A mutation in the NTD impaired m2G2445 formation, but rescued m7G2069 formation (Figure 3B). In contrast, the D568A mutation in the CTD did not rescue m7G2069 formation, but m2G2445 was efficiently formed in this mutant (Figure 3B). These observations suggested that the NTD and CTD of RlmKL encode an RlmL for m2G2445 formation and an RlmK for m7G2069 formation, respectively. Asn397 is located in the linker region between the NTD and CTD. Since the N397A mutant exhibited no m2G2445 formation but rescued m7G2069 formation, Asn397 should be considered part of RlmL. The other mutations, including D195A, R530A and D597A, did not affect methylation (data not shown).Figure 3.


Base methylations in the double-stranded RNA by a fused methyltransferase bearing unwinding activity.

Kimura S, Ikeuchi Y, Kitahara K, Sakaguchi Y, Suzuki T, Suzuki T - Nucleic Acids Res. (2011)

Complementation study of m7G2069 and m2G2445 formation by plasmid-encoded RlmKL and mutants. (A) Domain structure of E. coli RlmKL. RlmKL consists of two methyltransferase domains, COG0116 and COG1092. We designated the N-terminal domain (COG0116) as RlmL and the C-terminal domain (COG1092) as RlmK. Each domain has an Ado-Met-dependent methyltransferase motif, UPF0020 in the RlmL domain and Cons_hypoth 95 in the RlmK domain. A THUMP domain was found in the N-terminal region of the RlmL domain. (B) Complementation of the ΔrlmKL strain by introducing plasmid-encoded RlmKL or RlmKL mutants. On the left hand side, the domain structures and the mutation positions of RlmKL are shown. On the right hand side, mass chromatograms detecting the 16-mer fragment carrying m7G2069 (m/z 1706) and the hexamer fragment carrying m2G2445 (m/z 1024), respectively, are shown for the ΔrlmKL strain transformed with pRlmKL (top panels), pRlmKL N309A (second panels), pRlmKL N397A (third panels), pRlmK (fourth panels), pRlmKL D568A (fifth panels) and pRlmL (bottom panels). (C) Complementation of the E. coli ΔrlmKL strain by introducing N. meningtidis RlmL (NMB0445) or RlmK (NMB1367). Mass chromatograms on the right panels are the same as in (B).
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gkr1287-F3: Complementation study of m7G2069 and m2G2445 formation by plasmid-encoded RlmKL and mutants. (A) Domain structure of E. coli RlmKL. RlmKL consists of two methyltransferase domains, COG0116 and COG1092. We designated the N-terminal domain (COG0116) as RlmL and the C-terminal domain (COG1092) as RlmK. Each domain has an Ado-Met-dependent methyltransferase motif, UPF0020 in the RlmL domain and Cons_hypoth 95 in the RlmK domain. A THUMP domain was found in the N-terminal region of the RlmL domain. (B) Complementation of the ΔrlmKL strain by introducing plasmid-encoded RlmKL or RlmKL mutants. On the left hand side, the domain structures and the mutation positions of RlmKL are shown. On the right hand side, mass chromatograms detecting the 16-mer fragment carrying m7G2069 (m/z 1706) and the hexamer fragment carrying m2G2445 (m/z 1024), respectively, are shown for the ΔrlmKL strain transformed with pRlmKL (top panels), pRlmKL N309A (second panels), pRlmKL N397A (third panels), pRlmK (fourth panels), pRlmKL D568A (fifth panels) and pRlmL (bottom panels). (C) Complementation of the E. coli ΔrlmKL strain by introducing N. meningtidis RlmL (NMB0445) or RlmK (NMB1367). Mass chromatograms on the right panels are the same as in (B).
Mentions: Escherichia coli RlmKL is a fused methyltransferase consisting of 702 amino acid residues, containing a thiouridine synthases, methylases and PSUSs (THUMP) domain and two methyltransferase domains (Figure 3A). According to the cluster of orthologous group (COG) database (42), the N-terminal domain (NTD) and C-terminal domain (CTD) of RlmKL have been annotated as COG0116 and COG1092, respectively. Each domain has an Ado-Met-dependent methyltransferase motif (Figure 3A). The rlmKL gene is conserved in γ-proteobacteria (Supplementary Figure S1). However, in β-proteobacteria, Neisseria meningitidis bears COG0116 and COG1092 as separate proteins encoded at different genomic loci (Supplementary Figure S1). Since rlmKL appears to be responsible for two different methyl modifications, we hypothesized that each methyltransferase domain catalyzes the formation of one modification, either m7G2069 or m2G2445. To examine this possibility, we carried out complementation tests for the two methyl modifications in the ΔrlmKL strain by introducing various rlmKL mutants. We constructed rlmKL mutants in which conserved amino acid residues were substituted with Ala, including D195A, N309A, N397A, R530A, D568A and D597A. As a positive control, introduction of plasmid-encoded rlmKL into the ΔrlmKL strain restored both methyl modifications (Figure 3B). Subsequently, each of the rlmKL mutants was introduced into the ΔrlmKL strain, and formation of the methyl modifications was examined. The N309A mutation in the NTD impaired m2G2445 formation, but rescued m7G2069 formation (Figure 3B). In contrast, the D568A mutation in the CTD did not rescue m7G2069 formation, but m2G2445 was efficiently formed in this mutant (Figure 3B). These observations suggested that the NTD and CTD of RlmKL encode an RlmL for m2G2445 formation and an RlmK for m7G2069 formation, respectively. Asn397 is located in the linker region between the NTD and CTD. Since the N397A mutant exhibited no m2G2445 formation but rescued m7G2069 formation, Asn397 should be considered part of RlmL. The other mutations, including D195A, R530A and D597A, did not affect methylation (data not shown).Figure 3.

Bottom Line: The N-terminal RlmL activity for m(2)G2445 formation was significantly enhanced by the C-terminal RlmK.Moreover, RlmKL had an unwinding activity of Helix 74, facilitating cooperative methylations of m(7)G2069 and m(2)G2445 during biogenesis of 50S subunit.In fact, we observed that RlmKL was involved in the efficient assembly of 50S subunit in a mutant strain lacking an RNA helicase deaD.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry and Biotechnology, Graduate School of Engineering, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8656, Japan.

ABSTRACT
Modifications of rRNAs are clustered in functional regions of the ribosome. In Helix 74 of Escherichia coli 23S rRNA, guanosines at positions 2069 and 2445 are modified to 7-methylguanosine(m(7)G) and N(2)-methylguanosine(m(2)G), respectively. We searched for the gene responsible for m(7)G2069 formation, and identified rlmL, which encodes the methyltransferase for m(2)G2445, as responsible for the biogenesis of m(7)G2069. In vitro methylation of rRNA revealed that rlmL encodes a fused methyltransferase responsible for forming both m(7)G2069 and m(2)G2445. We renamed the gene rlmKL. The N-terminal RlmL activity for m(2)G2445 formation was significantly enhanced by the C-terminal RlmK. Moreover, RlmKL had an unwinding activity of Helix 74, facilitating cooperative methylations of m(7)G2069 and m(2)G2445 during biogenesis of 50S subunit. In fact, we observed that RlmKL was involved in the efficient assembly of 50S subunit in a mutant strain lacking an RNA helicase deaD.

Show MeSH
Related in: MedlinePlus