Analysis of 2'-O-methylated nucleosides and pseudouridines in ribosomal RNAs using DNAzymes.
Ribosomal RNAs (rRNAs) contain a large number of posttranscriptionally modified nucleosides, the physiological function of which is still unclear. The great majority of modifications in eukaryotes and archaea are 2'-O-ribose methylated nucleosides and pseudouridines. The current methods to identify rRNA modifications are difficult to perform and need expensive reagents. Here we report an easy method to detect 2'-O-ribose methylations using RNA-cleaving deoxyribozymes (DNAzymes) and demonstrate its application using rRNA of the yeast Saccharomyces cerevisiae. Using DNAzymes of the 10-23 type, we could show that cleavage at A(973) in the 18S rRNA or at G(1450) in the 25S rRNA from S. cerevisiae occurs only if the 2'-O-ribose methylations at these positions were missing. We also designed 8-17-DNAzymes for the detection of 2'-O-ribose methylations. This makes all 2'-O-ribose methylations accessible to the DNAzyme technique as variants of the 8-17-DNAzyme that together have the ability to cleave nearly any dinucleotide junction are known. Furthermore, we found that pseudouridine also decreases the DNAzyme cleavage efficiency at the adjacent phosphodiester bond and thus can also be identified with DNAzymes. The analysis using DNAzymes provides a new tool to easily identify ribose methylations in rRNAs and will help to unravel the physiological function of nucleotide modifications.
Buchhaupt
Markus
M
Center of Excellence, Macromolecular Complexes, Johann Wolfgang Goethe-University, Institute of Molecular Biosciences, 60438 Frankfurt/Main, Germany.
Peifer
Christian
C
Entian
Karl-Dieter
KD
D001483:Base Sequence; D021881:DNA, Catalytic / Q000378:metabolism*; D005838:Genotype; D007202:Indicators and Reagents; D008745:Methylation; D008969:Molecular Sequence Data; D009705:Nucleosides / Q000032:analysis*; D011560:Pseudouridine / Q000032:analysis*; D012323:RNA Processing, Post-Transcriptional; D012331:RNA, Fungal / Q000737:chemistry; D012335:RNA, Ribosomal / Q000737:chemistry*; D012441:Saccharomyces cerevisiae / Q000235:genetics
D021881:DNA, Catalytic; D007202:Indicators and Reagents; D009705:Nucleosides; D012331:RNA, Fungal; D012335:RNA, Ribosomal; D011560:Pseudouridine