InterPro domain: IPR004573

The rRNA small subunit methyltransferase B (RsmB) protein, often referred to as Fmu, has been demonstrated to methylate only C967 of the 16S ribosomal RNA and to produce only m5C at that position [ 1 ]. The structure of the E. coli protein has been determined [ 2 ]. It contains three subdomains which share structural homology to DNA m5C methyltransferases and two RNA binding protein families. The N-terminal sequence shares homology to another (noncatalytic) RNA binding protein, e.g. the ribosomal RNA antiterminator protein NusB ( IPR011605 ). The catalytic lobe of the N1 domain, comprises the conserved core identified in all of the putative RNA m5C MTase sequences. Although the N1 domain is structurally homologous to known RNA binding proteins, there is no clear sequence motif that defines its role in RNA binding and recognition. At the functional centre of the catalytic lobe is the MTase domain of Fmu (residues 232-429), which adopts a fold typical of known AdoMet-dependent methyltransferases. In spite of the lack of a conserved RNA binding motif in the N1 domain, the close association of the N1 and MTase domains suggest that any RNA bound in the active site of the MTase domain is likely to interact with the N1 domain.

RNA (C5-cytosine) methyltransferases (RCMTs) catalyse the transfer of a methyl group to the 5th carbon of a cytosine base in RNA sequences to produce C5-methylcytosine. RCMTs use the cofactor S-adenosyl-L-methionine (SAM) as a methyl donor [ 3 ]. The catalytic mechanism of RCMTs involves an attack by the thiolate of a Cys residue on position 6 of the target cytosine base to form a covalent link, thereby activating C5 for methyl-group transfer. Following the addition of the methyl group, a second Cys residue acts as a general base in the beta-elimination of the proton from the methylated cytosine ring. The free enzyme is restored and the methylated product is released [ 4 ].

Numerous putative RCMTs have been identified in archaea, bacteria and eukaryota [ 5 , 6 ]; most are predicted to be nuclear or nucleolar proteins [ 7 ]. The Escherichia coli Ribosomal RNA Small-subunit Methyltransferase Beta (RSMB) FMU (FirMicUtes) represents the first protein identified and characterised as a cytosine-specific RNA methyltransferase. RSMB was reported to catalyse the formation of C5-methylcytosine at position 967 of 16S rRNA [ 8 , 9 ].

A classification of RCMTs has been proposed on the basis of sequence similarity [ 9 ]. According to this classification, RCMTs are divided into 8 distinct subfamilies [ 9 ]. Recently, a new RCMT subfamily, termed RCMT9, was identified [ 9 ]. Members of the RCMT contain a core domain, responsible for the cytosine-specific RNA methyltransferase activity. This 'catalytic' domain adopts the Rossman fold for the accommodation of the cofactor SAM [ 9 ]. The RCMT subfamilies are also distinguished by N-terminal and C-terminal extensions, variable both in size and sequence [ 10 ].

1. Purification, cloning, and characterization of the 16S RNA m5C967 methyltransferase from Escherichia coli. Biochemistry 38, 1884-92
2. The first structure of an RNA m5C methyltransferase, Fmu, provides insight into catalytic mechanism and specific binding of RNA substrate. Structure 11, 1609-20
3. Many paths to methyltransfer: a chronicle of convergence. Trends Biochem. Sci. 28, 329-35
4. RNA methyltransferases utilize two cysteine residues in the formation of 5-methylcytosine. Biochemistry 41, 11218-25
5. Exposition of a family of RNA m(5)C methyltransferases from searching genomic and proteomic sequences. Nucleic Acids Res. 27, 3138-45
6. Phylogenetic analysis of the eukaryotic RNA (cytosine-5)-methyltransferases. Genomics 93, 350-7
7. 5-methylcytosine in RNA: detection, enzymatic formation and biological functions. Nucleic Acids Res. 38, 1415-30
8. Identification of the 16S rRNA m5C967 methyltransferase from Escherichia coli. Biochemistry 38, 4053-7
9. Sequence-structure-function studies of tRNA:m5C methyltransferase Trm4p and its relationship to DNA:m5C and RNA:m5U methyltransferases. Nucleic Acids Res. 32, 2453-63