Abstract
Guide box C/D small nucleolar RNAs (snoRNAs) catalyze 2'-O-methylation of ribosomal and small nuclear RNA. However, a large number of snoRNA in higher eukaryotes may promiscuously recognize other RNA species and 2'-O-methylate multiple targets. Here, we provide step-by-step guide for the fast and non-expensive analysis of the site-specific 2'-O-methylation using a well-established method employing short DNA oligonucleotides called DNAzymes. These DNA fragments contain catalytic sequences which cleave RNA at specific consensus positions, as well as variable homology arms directing DNAzyme to its RNA targets. DNAzyme activity is inhibited by 2-'O-methylation of the nucleotide adjacent to the cleavage site in the RNA. Thus, DNAzymes, limited only by the consensus of the cleaved sequence, are perfect tools for the quick analysis of snoRNA-mediated RNA 2'-O-methylation. We analyzed snoRNA snR13- and snR47-guided 2'-O-methylation of 25S ribosomal RNA in Saccharomyces cerevisiae to demonstrate the simplicity of the technique and to provide a detailed protocol for the DNAzyme-dependent assay.
Highlights
RNA modifications play important roles in the regulation of gene expression
Box C/D snR13 guides methylation at two positions in 25S ribosomal RNAs (rRNA), including adenine 2281 (Figure 2A). This nucleotide is followed by uracil, which constitutes the consensus dinucleotide (RY) cleavable by a 10-23 DNAzyme
RNA isolated from GAL1::SNR13 cells were incubated with [10-23] DNAzyme designed to cleave 25S rRNA at snR13-dependent site, in between nucleotides 2281 and 2282 (Figure 2C)
Summary
RNA modifications play important roles in the regulation of gene expression. RNA 2’-O-methylation and pseudouridylation, which are guided by box C/D and box H/ACA small nucleolar RNAs (snoRNAs) respectively, protect RNA from degradation and stabilize their higher-order structures[1,2,3]. A box C/D snoRNA-guided putative 2’-O-methylation site can be identified bioinformatically and confirmed experimentally by many techniques, including RNase H-directed cleavage, or site-specific and genome-wide methods, which employ reverse transcription in low nucleotides (dNTPs) concentration approach[8,9,10,11]. We provide a step-by-step protocol for the analysis of 2’-O-methylation of rRNA in Saccharomyces cerevisiae using [10-23] and [8,9,10,11,12,13,14,15,16,17] DNAzyme-dependent approaches[12,13] (Figure 1C) This protocol can be adapted for other organisms and RNA species and employed for the fast, preliminary or major analyses of site-specific RNA 2’-O-methylation. Any other yeast strain can be used for this analysis
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