Figure 1.
Identification of 18S rRNA molecules in wild type (WT) and Δsnr51 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 Δsnr51 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 Δsnr51 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.
Table 1.
DNAzyme sequences.