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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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In vitro reconstitution of m7G2069 and m2G2445 formation by RlmKL. (A) SDS–PAGE analysis of the purified recombinant RlmKL, RlmL(NTD) and RlmK(CTD) stained with coomassie Brilliant Blue R-250. (B) In vitro reconstitution of m7G2069 and m2G2445 by RlmKL, RlmL(NTD) or RlmK(CTD). Left and right panels show mass chromatograms detecting the triple-charged ions of the 16-mer fragment bearing m7G2069 (m/z 1706) and the double-charged ions of the hexamer fragment bearing m2G2445 (m/z 1024), respectively. Conditions for in vitro methylation are indicated in the right hand side. The hexamer fragment with m2G2445 produced by RNase A digestion of domain V is Gm2GGGAUp (m/z 1023, MW 2047). (C) Time-course of m2G2445 formation in domain V (transcript 1) catalyzed by 0.1 µM RlmL(NTD) (open circle), 0.1 µM RlmL(NTD) with 0.1 µM RlmK(CTD) (closed triangle), 0.1 µM RlmL(NTD) with 0.2 µM RlmK(CTD) (open triangle), 0.1 µM RlmL(NTD) with 0.4 µM RlmK(CTD) (closed square), 0.1 µM RlmL(NTD) with 0.8 µM RlmK(CTD) (open square) and 0.1 µM RlmKL (closed circle). (D) Time-course of m7G2069 formation in domain V (transcript 1) catalyzed by 0.1 µM RlmK(CTD) (open circle) and 0.1 µM RlmKL (closed circle). (E) Truncated RNA substrates. Open and closed circles in the secondary structures indicate the positions of G2069 and G2445, respectively. Detailed constructs for transcripts 3–7 are shown in Supplementary Figure S3. (F) Truncation of domain V (transcript 1) to identify the minimum substrate. The ratio of methylation (%) was calculated from the area of mass chromatograms for the methylated and unmethylated fragments at the end point of the reaction (see Materials and Methods). White and black bars represent the efficiency of m7G2069 and m2G2445 formation, respectively.
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gkr1287-F4: In vitro reconstitution of m7G2069 and m2G2445 formation by RlmKL. (A) SDS–PAGE analysis of the purified recombinant RlmKL, RlmL(NTD) and RlmK(CTD) stained with coomassie Brilliant Blue R-250. (B) In vitro reconstitution of m7G2069 and m2G2445 by RlmKL, RlmL(NTD) or RlmK(CTD). Left and right panels show mass chromatograms detecting the triple-charged ions of the 16-mer fragment bearing m7G2069 (m/z 1706) and the double-charged ions of the hexamer fragment bearing m2G2445 (m/z 1024), respectively. Conditions for in vitro methylation are indicated in the right hand side. The hexamer fragment with m2G2445 produced by RNase A digestion of domain V is Gm2GGGAUp (m/z 1023, MW 2047). (C) Time-course of m2G2445 formation in domain V (transcript 1) catalyzed by 0.1 µM RlmL(NTD) (open circle), 0.1 µM RlmL(NTD) with 0.1 µM RlmK(CTD) (closed triangle), 0.1 µM RlmL(NTD) with 0.2 µM RlmK(CTD) (open triangle), 0.1 µM RlmL(NTD) with 0.4 µM RlmK(CTD) (closed square), 0.1 µM RlmL(NTD) with 0.8 µM RlmK(CTD) (open square) and 0.1 µM RlmKL (closed circle). (D) Time-course of m7G2069 formation in domain V (transcript 1) catalyzed by 0.1 µM RlmK(CTD) (open circle) and 0.1 µM RlmKL (closed circle). (E) Truncated RNA substrates. Open and closed circles in the secondary structures indicate the positions of G2069 and G2445, respectively. Detailed constructs for transcripts 3–7 are shown in Supplementary Figure S3. (F) Truncation of domain V (transcript 1) to identify the minimum substrate. The ratio of methylation (%) was calculated from the area of mass chromatograms for the methylated and unmethylated fragments at the end point of the reaction (see Materials and Methods). White and black bars represent the efficiency of m7G2069 and m2G2445 formation, respectively.

Mentions: A reaction mixture (50 µl) consisting of 200 mM NH4OAc, 40 mM Tris–HCl (pH 7.5), 3 mM MgCl2, 6 mM β-mercaptoethanol, 1 mM Ado-Met, 0.2 µM 23S rRNA (or rRNA fragments) and 0.1 µM recombinant RlmKL [or RlmK(CTD), or RlmL(NTD)] was incubated at 37°C for 2 h. Substrate RNAs were recovered from aliquots of the reaction mixture by phenol–chloroform extraction and ethanol precipitation. RNA was digested by RNase T1 or RNase A and analyzed by LC/MS. To examine the time course of methylation (Figure 4C and D and Supplementary Figures S2 and S4), a 100-µl reaction mixture was prepared and 10-µl aliquots were taken at each time point and mixed with phenol–chloroform to stop the reaction. For preparing the m7G2069-containing domain V RNA, 50 pmol domain V RNA was incubated at 37°C with 200 pmol RlmK (CTD) in 100 µl reaction mixture for 2 h. Phenol–chloroform-extracted RNA was passed through NAP-5 column (GE healthcare) to remove Ado-Met (Ado-Hcy), and recovered by ethanol precipitation. In RNase T1 digested samples, we detected a 16-mer fragment bearing m7G2069 (Um7GAACCUUUACUAUAGp), or a 14-mer fragment without methylation (AACCUUUACUAUAGp). In RNase A digested samples, a tetramer fragment with (m7GAACp) or without (GAACp) m7G2069 and a hexamer fragment with (Gm2GGGAUp) or without (GGGGAUp) m2G2445 were detected. The methylated and unmethylated fragments were quantified by summing the areas of the mass chromatograms (±0.1 tolerance) of all detected ions. The frequency of methylation was calculated from the ratio of the areas of the mass chromatograms for the methylated and unmethylated fragments. For accurate quantification of both methylations, we generated a standard curve for the area ratio of the chromatograms against the ratio of the methylated fragments (Supplementary Figure S5). Using these curves, we could determine the exact values for methylation.


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)

In vitro reconstitution of m7G2069 and m2G2445 formation by RlmKL. (A) SDS–PAGE analysis of the purified recombinant RlmKL, RlmL(NTD) and RlmK(CTD) stained with coomassie Brilliant Blue R-250. (B) In vitro reconstitution of m7G2069 and m2G2445 by RlmKL, RlmL(NTD) or RlmK(CTD). Left and right panels show mass chromatograms detecting the triple-charged ions of the 16-mer fragment bearing m7G2069 (m/z 1706) and the double-charged ions of the hexamer fragment bearing m2G2445 (m/z 1024), respectively. Conditions for in vitro methylation are indicated in the right hand side. The hexamer fragment with m2G2445 produced by RNase A digestion of domain V is Gm2GGGAUp (m/z 1023, MW 2047). (C) Time-course of m2G2445 formation in domain V (transcript 1) catalyzed by 0.1 µM RlmL(NTD) (open circle), 0.1 µM RlmL(NTD) with 0.1 µM RlmK(CTD) (closed triangle), 0.1 µM RlmL(NTD) with 0.2 µM RlmK(CTD) (open triangle), 0.1 µM RlmL(NTD) with 0.4 µM RlmK(CTD) (closed square), 0.1 µM RlmL(NTD) with 0.8 µM RlmK(CTD) (open square) and 0.1 µM RlmKL (closed circle). (D) Time-course of m7G2069 formation in domain V (transcript 1) catalyzed by 0.1 µM RlmK(CTD) (open circle) and 0.1 µM RlmKL (closed circle). (E) Truncated RNA substrates. Open and closed circles in the secondary structures indicate the positions of G2069 and G2445, respectively. Detailed constructs for transcripts 3–7 are shown in Supplementary Figure S3. (F) Truncation of domain V (transcript 1) to identify the minimum substrate. The ratio of methylation (%) was calculated from the area of mass chromatograms for the methylated and unmethylated fragments at the end point of the reaction (see Materials and Methods). White and black bars represent the efficiency of m7G2069 and m2G2445 formation, respectively.
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gkr1287-F4: In vitro reconstitution of m7G2069 and m2G2445 formation by RlmKL. (A) SDS–PAGE analysis of the purified recombinant RlmKL, RlmL(NTD) and RlmK(CTD) stained with coomassie Brilliant Blue R-250. (B) In vitro reconstitution of m7G2069 and m2G2445 by RlmKL, RlmL(NTD) or RlmK(CTD). Left and right panels show mass chromatograms detecting the triple-charged ions of the 16-mer fragment bearing m7G2069 (m/z 1706) and the double-charged ions of the hexamer fragment bearing m2G2445 (m/z 1024), respectively. Conditions for in vitro methylation are indicated in the right hand side. The hexamer fragment with m2G2445 produced by RNase A digestion of domain V is Gm2GGGAUp (m/z 1023, MW 2047). (C) Time-course of m2G2445 formation in domain V (transcript 1) catalyzed by 0.1 µM RlmL(NTD) (open circle), 0.1 µM RlmL(NTD) with 0.1 µM RlmK(CTD) (closed triangle), 0.1 µM RlmL(NTD) with 0.2 µM RlmK(CTD) (open triangle), 0.1 µM RlmL(NTD) with 0.4 µM RlmK(CTD) (closed square), 0.1 µM RlmL(NTD) with 0.8 µM RlmK(CTD) (open square) and 0.1 µM RlmKL (closed circle). (D) Time-course of m7G2069 formation in domain V (transcript 1) catalyzed by 0.1 µM RlmK(CTD) (open circle) and 0.1 µM RlmKL (closed circle). (E) Truncated RNA substrates. Open and closed circles in the secondary structures indicate the positions of G2069 and G2445, respectively. Detailed constructs for transcripts 3–7 are shown in Supplementary Figure S3. (F) Truncation of domain V (transcript 1) to identify the minimum substrate. The ratio of methylation (%) was calculated from the area of mass chromatograms for the methylated and unmethylated fragments at the end point of the reaction (see Materials and Methods). White and black bars represent the efficiency of m7G2069 and m2G2445 formation, respectively.
Mentions: A reaction mixture (50 µl) consisting of 200 mM NH4OAc, 40 mM Tris–HCl (pH 7.5), 3 mM MgCl2, 6 mM β-mercaptoethanol, 1 mM Ado-Met, 0.2 µM 23S rRNA (or rRNA fragments) and 0.1 µM recombinant RlmKL [or RlmK(CTD), or RlmL(NTD)] was incubated at 37°C for 2 h. Substrate RNAs were recovered from aliquots of the reaction mixture by phenol–chloroform extraction and ethanol precipitation. RNA was digested by RNase T1 or RNase A and analyzed by LC/MS. To examine the time course of methylation (Figure 4C and D and Supplementary Figures S2 and S4), a 100-µl reaction mixture was prepared and 10-µl aliquots were taken at each time point and mixed with phenol–chloroform to stop the reaction. For preparing the m7G2069-containing domain V RNA, 50 pmol domain V RNA was incubated at 37°C with 200 pmol RlmK (CTD) in 100 µl reaction mixture for 2 h. Phenol–chloroform-extracted RNA was passed through NAP-5 column (GE healthcare) to remove Ado-Met (Ado-Hcy), and recovered by ethanol precipitation. In RNase T1 digested samples, we detected a 16-mer fragment bearing m7G2069 (Um7GAACCUUUACUAUAGp), or a 14-mer fragment without methylation (AACCUUUACUAUAGp). In RNase A digested samples, a tetramer fragment with (m7GAACp) or without (GAACp) m7G2069 and a hexamer fragment with (Gm2GGGAUp) or without (GGGGAUp) m2G2445 were detected. The methylated and unmethylated fragments were quantified by summing the areas of the mass chromatograms (±0.1 tolerance) of all detected ions. The frequency of methylation was calculated from the ratio of the areas of the mass chromatograms for the methylated and unmethylated fragments. For accurate quantification of both methylations, we generated a standard curve for the area ratio of the chromatograms against the ratio of the methylated fragments (Supplementary Figure S5). Using these curves, we could determine the exact values for methylation.

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