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 []. 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 [].Numerous putative RCMTs have been identified in archaea, bacteria and eukaryota [, ]; most are predicted to be nuclear or nucleolar proteins []. 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 [, ].A classification of RCMTs has been proposed on the basis of sequence similarity []. According to this classification, RCMTs are divided into 8 distinct subfamilies []. Recently, a new RCMT subfamily, termed RCMT9, was identified []. 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 []. The RCMT subfamilies are also distinguished by N-terminal and C-terminal extensions, variable both in size and sequence [].The prototypical member of the Nucleolar Protein 2 RCMT subfamily, the S.cerevisiae NOP2, is an essential nucleolar protein required for pre-rRNA processing and 60S ribosomal subunit assembly []that acts as a ribosomal RNA methyltransferase [, ]. Its human homologue, the proliferation-associated nucleolar antigen P120, is a promising tumour marker []. P120 has been demonstrated to be implicated in rRNA biogenesis [, ], and is also proposed to act as an rRNA methyltransferase [].
This characteristic repeat of proliferating cell nuclear antigen P120 is found in three copies []. This repeat can be found in the human NOP2 protein, a probable 28S rRNA (cytosine(4447)-C(5))-methyltransferase.
The C-terminal catalytic domain of ribosomal RNA cysteine methyltransferases is highly conserved in archaeal, bacterial and eukaryotic proteins [], such as ribosomal RNA methyltransferase B (RsmB, Sun, Fmu) and Nop2. Escherichia coli RsmB methylates cytosine C967 in 16S rRNA []. Nop2 methylates cytosine C2870 in the 25S rRNA of S. cerevisiae []and is critical for 60S biogenesis [].
