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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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ycbY (rlmKL) is responsible for both m7G2069 and m2G2445 formation. (A) LC/MS analyses of RNase T1 (left panels) and RNase A (right panels) digests of rRNAs from the wild-type (Top row of panels), genome-deletion strain OCR55 (Second row), ΔycbY (rlmKL) strain (Third row) and ΔycbY (rlmKL) strain carrying pRlmKL (Bottom row). Left and right panels show mass chromatograms detecting the triply-charged ions of the 16-mer fragment bearing m7G2069 (m/z 1706) and the doubly charged ions of the hexamer fragment bearing m2G2445 (m/z 1024), respectively. (B) Collision-induced dissociation (CID) spectra of RNA fragments bearing m7G2069 (left panel) and m2G2445 (right panel). The six-charged ion (m/z 852.4) of the 16-mer fragment carrying m7G2069 and doubly charged ion (m/z 1023.7) of the hexamer fragment carrying m2G2445 were used as parent ions for CID. The sequences were confirmed by assignment of the product ions. The nomenclatures for product ions of nucleic acids are as suggested in the literature (41).
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gkr1287-F2: ycbY (rlmKL) is responsible for both m7G2069 and m2G2445 formation. (A) LC/MS analyses of RNase T1 (left panels) and RNase A (right panels) digests of rRNAs from the wild-type (Top row of panels), genome-deletion strain OCR55 (Second row), ΔycbY (rlmKL) strain (Third row) and ΔycbY (rlmKL) strain carrying pRlmKL (Bottom row). Left and right panels show mass chromatograms detecting the triply-charged ions of the 16-mer fragment bearing m7G2069 (m/z 1706) and the doubly charged ions of the hexamer fragment bearing m2G2445 (m/z 1024), respectively. (B) Collision-induced dissociation (CID) spectra of RNA fragments bearing m7G2069 (left panel) and m2G2445 (right panel). The six-charged ion (m/z 852.4) of the 16-mer fragment carrying m7G2069 and doubly charged ion (m/z 1023.7) of the hexamer fragment carrying m2G2445 were used as parent ions for CID. The sequences were confirmed by assignment of the product ions. The nomenclatures for product ions of nucleic acids are as suggested in the literature (41).

Mentions: To identify gene products responsible for rRNA modifications in E. coli, we employed a reverse genetic approach combined with RNA MS (ribonucleome analysis) for genome-wide screening in E. coli (20,28). Total RNA, consisting primarily of rRNAs and mRNAs, was prepared from 94 genomic-deletion strains of E. coli (38), each of which lacks about 20 kb (∼20 genes) (see Supplementary Data). Thus, this analysis covered almost 2000 genes, ∼40% of the total 4762 genes of E. coli. Total RNA was digested into fragments by base-specific ribonucleases (RNase T1 or RNase A) and the digests were analyzed by capillary LC coupled with nano ESI LC/MS. In these complex mixtures of RNA fragments, we can detect RNA fragments containing modified nucleoside(s) as multiply-charged negative ions by monitoring the specific mass-to-charge ratio (m/z) value. Using both RNase T1 and RNase A, we can detect all modifications to be analyzed. In wild-type E. coli, we clearly detected the triple-charged negative ion (m/z 1706) of a 16-mer fragment including m7G2069 (Um7GAACCUUUACUAUAGp; MW 5120) produced by RNase T1 digestion, and the double-charged negative ion (m/z 1024) of a hexamer fragment including m2G2445 (Gm2GGGADp; MW 2049) produced by RNase A digestion (Figures 1B and 2A). Each fragment was analyzed by collision-induced dissociation (CID) by MS/MS to confirm the exact position of the modification (Figure 2B). When we analyzed RNA fragments from the deletion strain OCR55 (ΔpncB-rmf), the 16-mer fragment bearing m7G2069 was absent (Figure 2A). If 7-methylation of G does not occur at position 2069, an unmodified 14-mer fragment (AACCUUUACUAUAGp; m/z 1485, MW 4454) should be detected. In fact, we clearly detected this 14-mer fragment from the OCR55 strain (data not shown), confirming the absence of m7G2069. These data indicated that a gene responsible for m7G2069 formation resides in this deleted region. Since rlmL/ycbY is also localized in this region, no hexamer fragment bearing m2G2445 was observed (Figure 2A). To identify the gene responsible for m7G2069 formation, we analyzed 23 single gene knockout strains (KO collections), each of which lacks one gene in the deleted region of OCR55. The ΔrlmL/ycbY strain was found to lack the 16-mer fragment bearing m7G2069 (Figure 2A). To our surprise, rlmL/ycbY gene encodes a known methyltransferase, RlmL, which is responsible for m2G2445 formation (34) and in fact, no hexamer fragment bearing m2G2445 was detected in this strain (Figure 2A). When a plasmid encoding rlmL/ycbY was introduced into the ΔrlmL/ycbY strain, both m7G2069 and m2G2445 were detected in the total RNA (Figure 2A). These data clearly demonstrated that rlmL/ycbY is responsible not only for m2G2445 formation, but also for m7G2069 formation. Therefore, we renamed the gene rlmKL according to the preferred nomenclature (39,40).Figure 2.


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)

ycbY (rlmKL) is responsible for both m7G2069 and m2G2445 formation. (A) LC/MS analyses of RNase T1 (left panels) and RNase A (right panels) digests of rRNAs from the wild-type (Top row of panels), genome-deletion strain OCR55 (Second row), ΔycbY (rlmKL) strain (Third row) and ΔycbY (rlmKL) strain carrying pRlmKL (Bottom row). Left and right panels show mass chromatograms detecting the triply-charged ions of the 16-mer fragment bearing m7G2069 (m/z 1706) and the doubly charged ions of the hexamer fragment bearing m2G2445 (m/z 1024), respectively. (B) Collision-induced dissociation (CID) spectra of RNA fragments bearing m7G2069 (left panel) and m2G2445 (right panel). The six-charged ion (m/z 852.4) of the 16-mer fragment carrying m7G2069 and doubly charged ion (m/z 1023.7) of the hexamer fragment carrying m2G2445 were used as parent ions for CID. The sequences were confirmed by assignment of the product ions. The nomenclatures for product ions of nucleic acids are as suggested in the literature (41).
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Related In: Results  -  Collection

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gkr1287-F2: ycbY (rlmKL) is responsible for both m7G2069 and m2G2445 formation. (A) LC/MS analyses of RNase T1 (left panels) and RNase A (right panels) digests of rRNAs from the wild-type (Top row of panels), genome-deletion strain OCR55 (Second row), ΔycbY (rlmKL) strain (Third row) and ΔycbY (rlmKL) strain carrying pRlmKL (Bottom row). Left and right panels show mass chromatograms detecting the triply-charged ions of the 16-mer fragment bearing m7G2069 (m/z 1706) and the doubly charged ions of the hexamer fragment bearing m2G2445 (m/z 1024), respectively. (B) Collision-induced dissociation (CID) spectra of RNA fragments bearing m7G2069 (left panel) and m2G2445 (right panel). The six-charged ion (m/z 852.4) of the 16-mer fragment carrying m7G2069 and doubly charged ion (m/z 1023.7) of the hexamer fragment carrying m2G2445 were used as parent ions for CID. The sequences were confirmed by assignment of the product ions. The nomenclatures for product ions of nucleic acids are as suggested in the literature (41).
Mentions: To identify gene products responsible for rRNA modifications in E. coli, we employed a reverse genetic approach combined with RNA MS (ribonucleome analysis) for genome-wide screening in E. coli (20,28). Total RNA, consisting primarily of rRNAs and mRNAs, was prepared from 94 genomic-deletion strains of E. coli (38), each of which lacks about 20 kb (∼20 genes) (see Supplementary Data). Thus, this analysis covered almost 2000 genes, ∼40% of the total 4762 genes of E. coli. Total RNA was digested into fragments by base-specific ribonucleases (RNase T1 or RNase A) and the digests were analyzed by capillary LC coupled with nano ESI LC/MS. In these complex mixtures of RNA fragments, we can detect RNA fragments containing modified nucleoside(s) as multiply-charged negative ions by monitoring the specific mass-to-charge ratio (m/z) value. Using both RNase T1 and RNase A, we can detect all modifications to be analyzed. In wild-type E. coli, we clearly detected the triple-charged negative ion (m/z 1706) of a 16-mer fragment including m7G2069 (Um7GAACCUUUACUAUAGp; MW 5120) produced by RNase T1 digestion, and the double-charged negative ion (m/z 1024) of a hexamer fragment including m2G2445 (Gm2GGGADp; MW 2049) produced by RNase A digestion (Figures 1B and 2A). Each fragment was analyzed by collision-induced dissociation (CID) by MS/MS to confirm the exact position of the modification (Figure 2B). When we analyzed RNA fragments from the deletion strain OCR55 (ΔpncB-rmf), the 16-mer fragment bearing m7G2069 was absent (Figure 2A). If 7-methylation of G does not occur at position 2069, an unmodified 14-mer fragment (AACCUUUACUAUAGp; m/z 1485, MW 4454) should be detected. In fact, we clearly detected this 14-mer fragment from the OCR55 strain (data not shown), confirming the absence of m7G2069. These data indicated that a gene responsible for m7G2069 formation resides in this deleted region. Since rlmL/ycbY is also localized in this region, no hexamer fragment bearing m2G2445 was observed (Figure 2A). To identify the gene responsible for m7G2069 formation, we analyzed 23 single gene knockout strains (KO collections), each of which lacks one gene in the deleted region of OCR55. The ΔrlmL/ycbY strain was found to lack the 16-mer fragment bearing m7G2069 (Figure 2A). To our surprise, rlmL/ycbY gene encodes a known methyltransferase, RlmL, which is responsible for m2G2445 formation (34) and in fact, no hexamer fragment bearing m2G2445 was detected in this strain (Figure 2A). When a plasmid encoding rlmL/ycbY was introduced into the ΔrlmL/ycbY strain, both m7G2069 and m2G2445 were detected in the total RNA (Figure 2A). These data clearly demonstrated that rlmL/ycbY is responsible not only for m2G2445 formation, but also for m7G2069 formation. Therefore, we renamed the gene rlmKL according to the preferred nomenclature (39,40).Figure 2.

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