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Many viruses from the realm Riboviria infecting eukaryotic hosts encode protein domains with sequence similarity to S-adenosylmethionine-dependent methyltransferases. These protein domains are thought to be involved in methylation of the 5'-terminal cap structures in virus mRNAs. Some methyltransferase-like domains of Riboviria are homologous to the widespread cellular FtsJ/RrmJ-like methyltransferases involved in modification of cellular RNAs; other methyltransferases, found in a subset of positive-strand RNA viruses, have been assigned to a separate "Sindbis-like" family; and coronavirus-specific Nsp13/14-like methyltransferases appeared to be different from both those classes. The representative structures of proteins from all three groups belong to a specific variety of the Rossmann fold with a seven-stranded β-sheet, but it was unclear whether this structural similarity extends to the level of conserved sequence signatures. Here I survey methyltransferases in Riboviria and derive a joint sequence alignment model that covers all groups of virus methyltransferases and subsumes the previously defined conserved sequence motifs. Analysis of the spatial structures indicates that two highly conserved residues, a lysine and an aspartate, frequently contact a water molecule, which is located in the enzyme active center next to the methyl group of S-adenosylmethionine cofactor and could play a key role in the catalytic mechanism of the enzyme. Phylogenetic evidence indicates a likely origin of all methyltransferases of Riboviria from cellular RrmJ-like enzymes and their rapid divergence with infrequent horizontal transfer between distantly related viruses.

Ribosome biogenesis is a complex process, and dozens of factors are required to facilitate and regulate the subunit assembly in bacteria. The 2'-O-methylation of U2552 in 23S rRNA by methyltransferase RrmJ is a crucial step in late-stage assembly of the 50S subunit. Its absence results in severe growth defect and marked accumulation of pre50S assembly intermediates. In the present work, we employed cryoelectron microscopy to characterize a set of late-stage pre50S particles isolated from an Escherichia coli Δ rrmJ strain. These assembly intermediates (solved at 3.2 to 3.8 Å resolution) define a collection of late-stage particles on a progressive assembly pathway. Apart from the absence of L16, L35, and L36, major structural differences between these intermediates and the mature 50S subunit are clustered near the peptidyl transferase center, such as H38, H68-71, and H89-93. In addition, the ribosomal A-loop of the mature 50S subunit from Δ rrmJ strain displays large local flexibility on nucleotides next to unmethylated U2552. Fast kinetics-based biochemical assays demonstrate that the Δ rrmJ 50S subunit is only 50% active and two times slower than the WT 50S subunit in rapid subunit association. While the Δ rrmJ 70S ribosomes show no defect in peptide bond formation, peptide release, and ribosome recycling, they translocate with 20% slower rate than the WT ribosomes in each round of elongation. These defects amplify during synthesis of the full-length proteins and cause overall defect in protein synthesis. In conclusion, our data reveal the molecular roles of U2552 methylation in both ribosome biogenesis and protein translation., Competing Interests: The authors declare no competing interest.