Identification of a novel methyltransferase, Bmt2, responsible for the N-1-methyl-adenosine base modification of 25S rRNA in Saccharomyces cerevisiae

Abstract

The 25S rRNA of yeast contains several base modifications in the functionally important regions. The enzymes responsible for most of these base modifications remained unknown. Recently, we identified Rrp8 as a methyltransferase involved in m1A645 modification of 25S rRNA. Here, we discovered a previously uncharacterized gene YBR141C to be responsible for second m1A2142 modification of helix 65 of 25S rRNA. The gene was identified by reversed phase–HPLC screening of all deletion mutants of putative RNA methyltransferase and was confirmed by gene complementation and phenotypic characterization. Because of the function of its encoded protein, YBR141C was named BMT2 (base methyltransferase of 25S RNA). Helix 65 belongs to domain IV, which accounts for most of the intersubunit surface of the large subunit. The 3D structure prediction of Bmt2 supported it to be an Ado Met methyltransferase belonging to Rossmann fold superfamily. In addition, we demonstrated that the substitution of G180R in the S-adenosyl-l-methionine–binding motif drastically reduces the catalytic function of the protein in vivo. Furthermore, we analysed the significance of m1A2142 modification in ribosome synthesis and translation. Intriguingly, the loss of m1A2142 modification confers anisomycin and peroxide sensitivity to the cells. Our results underline the importance of RNA modifications in cellular physiology.

Structural and functional studies of Bud23–Trm112 reveal 18S rRNA N 7 -G1575 methylation occurs on late 40S precursor ribosomes

“…The six enzymes involved, Bmt2, Bmt5, Bmt6, Nop2, Rcm1, and Rrp8, have only been identified recently (12)(13)(14)(15) (8,17,18). In contrast to the majority of large subunit MTases, deletion of genes encoding modification enzymes specific to the small ribosomal…”

Role of RNA modifications in brain and behavior

“…15,16 m 1 A is found in all regions of transcripts including the coding sequence (CDS), as well as 5’ and 3’ untranslated regions (UTRs). 17,18 mRNA molecules have also been found to contain m 1 A although its function is less well-known.…”

NML-mediated rRNA base methylation links ribosomal subunit formation to cell proliferation in a p53-dependent manner

“…Primer extension was performed as per previously described protocols (Cozen et al, 2015;Sharma et al, 2013) with minor modifications. One micromole of DNA primer was 32 P-5′-terminally labeled by incubation in a final volume of 25 μl with 15 μCi γ-[…”

“…Base methylation is considered to expand the structural repertoire of RNA, facilitating base stacking by increasing hydrophobicity and adjusting steric hindrance (Ishitani et al, 2008). To date, base methylation of rRNA and the genes responsible for it (Bud23, Rrp8, Nop2, Rcm1, Bmt2, Bmt5 and Bmt6) have been reported in budding yeast (Gigova et al, 2014;Peifer et al, 2013;Sharma et al, 2013;White et al, 2008). A very recent report has shown that yeast and worm homologs of human NSUN5 methylate C2278 in 25S rRNA of Saccharomyces cerevisiae and C2381 in 26S rRNA of Caenorhabditis elegans; in addition, the reduction in the levels of NSUN5 homologs decreases translational fidelity in yeast and increases the lifespan of S. cerevisiae, C. elegans and Drosophila melanogaster (Schosserer et al, 2015).…”