Yeast Nop2 and Rcm1 methylate C2870 and C2278 of the 25S rRNA, respectively

Abstract

Yeast 25S rRNA was reported to contain a single cytosine methylation (m5C). In the present study using a combination of RP-HPLC, mung bean nuclease assay and rRNA mutagenesis, we discovered that instead of one, yeast contains two m5C residues at position 2278 and 2870. Furthermore, we identified and characterized two putative methyltransferases, Rcm1 and Nop2 to be responsible for these two cytosine methylations, respectively. Both proteins are highly conserved, which correlates with the presence of two m5C residues at identical positions in higher eukaryotes, including humans. The human homolog of yeast Nop2, p120 has been discovered to be upregulated in various cancer tissues, whereas the human homolog of Rcm1, NSUN5 is completely deleted in the William's-Beuren Syndrome. The substrates and function of both human homologs remained unknown. In the present study, we also provide insights into the significance of these two m5C residues. The loss of m5C2278 results in anisomycin hypersensitivity, whereas the loss of m5C2870 affects ribosome synthesis and processing. Establishing the locations and enzymes in yeast will not only help identifying the function of their homologs in higher organisms, but will also enable understanding the role of these modifications in ribosome function and architecture.

A cluster of methylations in the domain IV of 25S rRNA is required for ribosome stability

“…In a recent report (Sharma et al 2013), polysome profiling of yeast lacking Rcm1p did not produce such an obvious "shoulder." However, Sharma et al (2013) used higher sucrose concentrations in their gradient centrifugation that probably resulted in a more compressed profile with such a shoulder far less obvious.…”

“…We did not observe anisomycin sensitivity in our rcm1Δ strain. Interestingly, Sharma et al (2013) reported recently that yeast strains lacking Rcm1p were also anisomycin sensitive. This discrepancy could be due to different genetic backgrounds of the parental strains used in each study and/or different experimental setups used to assay the sensitivity.…”

“…This discrepancy could be due to different genetic backgrounds of the parental strains used in each study and/or different experimental setups used to assay the sensitivity. Moreover, according to the Materials and Methods section, Sharma et al (2013) used higher concentrations of anisomycin (20 μg/mL) than were used in this study (5 μg/mL).…”

“…The cytosine-5 methyltransferase for C2278 in yeast was unknown prior to this study. During the writing of this manuscript, the identification of Rcm1p as a methyltransferase for C2278 has been independently reported (Sharma et al 2013).…”

Statistically robust methylation calling for whole-transcriptome bisulfite sequencing reveals distinct methylation patterns for mouse RNAs

“…In this context, it has been shown that m5C modification of tRNA plays an important role in tRNA stability and in the regulation of translational fidelity (Schaefer et al 2010;Tuorto et al 2012Tuorto et al , 2015. Furthermore, m5C is also a widely conserved modification of rRNA, where it is implied in the quality control of ribosome biogenesis (Sharma et al 2013;Bourgeois et al 2015;Schosserer et al 2015). These processes have been associated with a variety of human diseases (Blanco and Frye 2014).…”