Partial methylation at Am100 in 18S rRNA of baker's yeast reveals ribosome heterogeneity on the level of eukaryotic rRNA modification - PubMed (original) (raw)

Partial methylation at Am100 in 18S rRNA of baker's yeast reveals ribosome heterogeneity on the level of eukaryotic rRNA modification

Markus Buchhaupt et al. PLoS One. 2014.

Abstract

Ribosome heterogeneity is of increasing biological significance and several examples have been described for multicellular and single cells organisms. In here we show for the first time a variation in ribose methylation within the 18S rRNA of Saccharomyces cerevisiae. Using RNA-cleaving DNAzymes, we could specifically demonstrate that a significant amount of S. cerevisiae ribosomes are not methylated at 2'-O-ribose of A100 residue in the 18S rRNA. Furthermore, using LC-UV-MS/MS of a respective 18S rRNA fragment, we could not only corroborate the partial methylation at A100, but could also quantify the methylated versus non-methylated A100 residue. Here, we exhibit that only 68% of A100 in the 18S rRNA of S.cerevisiae are methylated at 2'-O ribose sugar. Polysomes also contain a similar heterogeneity for methylated Am100, which shows that 40S ribosome subunits with and without Am100 participate in translation. Introduction of a multicopy plasmid containing the corresponding methylation guide snoRNA gene SNR51 led to an increased A100 methylation, suggesting the cellular snR51 level to limit the extent of this modification. Partial rRNA modification demonstrates a new level of ribosome heterogeneity in eukaryotic cells that might have substantial impact on regulation and fine-tuning of the translation process.

PubMed Disclaimer

Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1. Identification of 18S rRNA molecules in wild type (WT) and Δ_snr51_ that lack the 2′-_O_-ribose methyl group at nucleotide A100.

A) Detection of 18S rRNA molecules lacking the Am100 modification by DNAzyme cleavage. Total RNA from wild type and Δ_snr51_ cells grown in YEPD medium to exponential phase was incubated with or without the DNAzyme 10-23-snR51-A100 and afterwards analyzed by gel analysis. The RNA band marked with an asterisk represents the large 18S rRNA fragment after cleavage at nucleotide A100. The 100 nucleotide fragment ran out of the gel. B) Schematic illustration of DNAzyme 10–23-snR51-A100 binding to its target site in 18S rRNA. The cleavage site 3′ to A100 is marked with an arrow. The resulting fragments have lengths of 100 nt and 1700 nt.

Figure 2. LC-UV-MS/MS analysis of the isolated rRNA fragment and calibration samples.

(A) Schematic diagram for the mung bean nuclease method, used here to isolate the 18S rRNA fragment containing Am100 residue for subsequent mass spectrometry analysis. (B) UV chromatograms of all 4 samples and peak areas. In red, the sample containing 100% guanosine and 10% 2′-_O_-methyladenosine is shown. In green and blue the respective 50% and 100% Am turnover samples are shown. The black chromatogram shows the guanosine peak of the rRNA fragment. (C) Overlay of MS/MS chromatograms for 2′-_O_-methyladenosine. The methylation extent of the rRNA fragment was found to be 68% as could be seen in comparison to the calibration samples.

Figure 3. Comparison of 18S rRNA molecules lacking the 2′-_O_-ribose methyl group at nucleotide A100 in polysomal RNA and total RNA.

Polysomal RNA and total RNA isolated from wild type cells that were grown in YEPD medium to exponential phase was incubated with or without the DNAzyme 10–23-snR51-A100 and afterwards analyzed by gel analysis. The RNA band marked with an asterisk represents the large 18S rRNA fragment after cleavage at nucleotide A100.

Figure 4. Investigation of changes in Am100 modification extent in 18S rRNA after introduction of a multicopy plasmid containing SNR51.

Total RNA isolated from wild type and Δ_snr51_ cells grown in synthetic medium to exponential phase containing no plasmid, the plasmid pRS426 or the plasmid pRS426-Cluster3 was incubated with the DNAzyme 10–23-snR51-A100 and afterwards analyzed by gel analysis. The RNA band marked with an asterisk represents the large 18S rRNA fragment after cleavage at nucleotide A100.

References

1. Anger AM, Armache JP, Berninghausen O, Habeck M, Subklewe M, et al. (2013) Structures of the human and Drosophila 80S ribosome. Nature 497: 80–85. - PubMed
1. Ramagopal S (1992) Are eukaryotic ribosomes heterogeneous? Affirmations on the horizon. Biochem Cell Biol 70: 269–272. - PubMed
1. Delaunay J, Mathieu C, Schapira G (1972) Eukaryotic ribosomal proteins. Interspecific and intraspecific comparisons by two-dimensional polyacrylamide-gel electrophoresis. Eur J Biochem 31: 561–564. - PubMed
1. Lambertsson AG (1975) The ribosomal proteins of Drosophila melanogaster. IV. Characterization by two-dimensional gel electrophoresis of the ribosomal proteins from nine postembryonic developmental stages. Mol Gen Genet 139: 133–144. - PubMed
1. Gunderson JH, Sogin ML, Wollett G, Hollingdale M, de la Cruz VF, et al. (1987) Structurally distinct, stage-specific ribosomes occur in Plasmodium . Science 238: 933–937. - PubMed

Partial methylation at Am100 in 18S rRNA of baker's yeast reveals ribosome heterogeneity on the level of eukaryotic rRNA modification - PubMed (original) (raw)