Here we present a protocol for DNAzyme-dependent cleavage of RNA. This enables fast and site-dependent analysis of RNA 2’-O-methylation. This approach can be used for the preliminary or major assessment of snoRNA activity.
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.
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 structures1,2,3. SnoRNA targets have been identified mainly in ribosomal RNAs (rRNA) and small nuclear RNAs (snRNAs). However, in higher eukaryotes, there are potentially hundreds of snoRNA with no assigned functions and some of them may recognize multiple RNAs1,4,5,6,7. Therefore, methods which allow for the identification and analysis of snoRNA-guided modifications are important tools in uncovering mechanisms governing cellular processes.
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 approach8,9,10,11. These techniques are very sensitive but also laborious and expensive, therefore, may not be suitable for the initial or quick testing. One of the simplest and low-cost methods to identify 2’-O-methylation sites is DNAzyme-dependent RNA cleavage12. DNAzymes are short, single-stranded and catalytically active DNA molecules capable of endonucleolytic cleavage of RNA at specific positions. They consist of a conserved and catalytically active core sequence and 5’ and 3’ binding arms composed of variable sequences designed to hybridize by Watson-Crick base-pairing to the RNA target (Figure 1). Thus, the 5’ and 3’ arms deliver the catalytic sequence to the specific RNA site. DNAzyme-dependent cleavage is inhibited by 2’-O-methylation of the nucleotide positioned directly upstream of the cleavage site12,13. This makes DNAzymes very practical tools for the analysis of putative or known RNA 2’-O-methylation sites.
Two types of DNAzymes are used for RNA modifications analyses12. The active sequence of 10-23 DNAzyme (Figure 1A) consists of 15 nucleotides (5’RGGCTAGCTACAACGA3’) which form a loop around the targeted RNA purine-pyrimidine (RY) dinucleotide and catalyze the cleavage between these two nucleotides. The RNA purine (R) is not base-paired with the DNAzyme and the 2’-O-methylation presents on the DNAzyme inhibits the cleavage. The binding arms of 10-23 DNAzymes are usually 10-15 nucleotides long. The second DNAzyme class, 8-17 DNAzymes (Figure 1B) contain 14-nucleotide catalytic sequence (5’TCCGAGCCGGACGA3’). Nucleotides C2, C3 and G4 pair with C9 G10 and G11 forming a short stem-loop structure. 8-17 DNAzymes cleave RNA upstream of any guanine that is imperfectly paired with the first thymine from the DNAzyme active sequence. The RNA nucleotide upstream of the guanine is not base-paired with DNAzyme and its 2’-O-methylation impairs the cleavage. 8-17 DNAzymes require longer homology arms of around 20 nucleotides to direct DNAzyme to its specific sequence.
Here, we provide a step-by-step protocol for the analysis of 2’-O-methylation of rRNA in Saccharomyces cerevisiae using 10-23 and 8-17 DNAzyme-dependent approaches12,13 (Figure 1C). This protocol can be easily adapted for other organisms and RNA species and employed for the fast, preliminary or major analyses of site-specific RNA 2’-O-methylation.
1. Strains, Media, and Buffer Recipes
2. DNAzyme Design
3. S. cerevisiae Growth Conditions
NOTE: S. cerevisiae BY4741 strain derivatives were used, in which the expression of either SNR13 or SNR47 snoRNA is driven from the inducible GAL1 promoter. In order to induce or inhibit their synthesis, grow cells on medium containing galactose (GAL1-dependent transcription on) or glucose (GAL1-dependent transcription off). As a control, use the wild type strain (BY4741) grown either on galactose or glucose.
4. RNA Isolation15
NOTE: Use the most appropriate method to isolate RNA. For yeast S. cerevisiae, hot-phenol RNA extraction can be used.
5. DNAzyme Digestion
6. RNA Electrophoresis
The utility of the DNAzyme-dependent cleavage in the analysis of rRNA modifications has been shown recently in the context of snoRNAs maturation13. The DNAzyme-dependent assay was used to show that lack of 5’-end pre-snoRNA processing affects 2’-O-methylation levels of 25S and 18S rRNA in S. cerevisiae13.
Here, we used an inducible snoRNA transcription system to demonstrate the effectiveness and simplicity of the technique. Box C/D snR13 guides methylation at two positions in 25S rRNA, including adenine 2281 (Figure 2A). This nucleotide is followed by uracil, which constitutes the consensus dinucleotide (RY) cleavable by a 10-23 DNAzyme. Box C/D snR47 also guides methylation of two nucleotides in 25S rRNA (Figure 2B). The adenine in position 2220 is followed by a guanine residue and this dinucleotide can be cleaved by an 8-17 DNAzyme. In order to induce or inhibit synthesis of either snR13 or snR47 snoRNA, we inserted the inducible GAL1 promoter upstream of either SNR13 or SNR47 genes and cultivated cells in medium containing galactose (GAL1-dependent transcription on) or glucose (GAL1-dependent transcription off). Next, 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). RNA from GAL1::SNR47 strain was treated with 8-17 DNAzyme targeting snR47-dependent site in between nucleotides 2220 and 2221 (Figure 2D). As a control, RNA from the wild-type BY4741 strain growing on either galactose or glucose was incubated with both DNAzymes. Electrophoresis of the DNAzyme-treated RNA revealed that 25S rRNA extracted from GAL1::SNR13 and GAL1::SNR47 strains growing on galactose (GAL) remained intact (Figure 3A,B; lanes 3). In contrast, RNA isolated from GAL1::SNR13 and GAL1::SNR47 cells growing on glucose (GLC) was digested by respective DNAzymes (Figure 3A,B; lanes 4). In both cases, the 25S rRNA band decreased and 5’ and 3’ cut-off cleavage products (A and B) were observed. This indicates that in GAL1::SNR13 and GAL1::SNR47 strains, 25S rRNA was 2’-O-methylated at snR13- or snR47-guided sites when galactose was used as a carbon source and these snoRNA were expressed. The lack of 25S rRNA methylation when snR13 or snR47 expression was shut off on glucose allowed for DNAzyme-dependent cleavage. No RNA digestion was observed for wild-type samples (Figure 3A,B; lanes 1 and 2), as the expression of snR13 and snR47 is galactose/glucose-independent in this strain. Therefore, rRNA was normally methylated and so resistant to DNAzymes activity.
Overall, our experiment shows that the cleavage activity of 10-23 (Figure 3A) and 8-17 (Figure 3B) DNAzymes correlated with the absence of box C/D snR13 or snR47, clearly indicating that these snoRNA are responsible for 25S rRNA 2’-O-methylation at particular sites.
Figure 1: DNAzymes and their RNA substrates. (A) 10-23 DNAzymes cleave a purine-pyrimidine (RY) RNA dinucleotide. R in the RNA is not paired with DNAzyme, while Y is complementary to the R base in the DNAzyme. Methylation of the purine (R) in RNA suppresses DNAzyme-dependent cleavage. (B) 8-17 DNAzymes cleave RNA upstream of guanine that is imperfectly paired with the first thymine in the DNAzyme catalytic sequence. The nucleotide preceding guanine is not paired and its methylation protects from DNAzyme-dependent cleavage. RNA is shown in grey (apart from the methylation site), DNAzyme is shown in purple. N = any nucleotide, R = purine: adenine or guanine, Y = pyrimidine: cytosine or uracil; CH3- denotes RNA methylation. Base pairing within the DNAzyme active sequences is marked by dotted lines. A blue lightning bolt marks the cleavage site. (C) A flowchart showing the steps of a DNAzyme-dependent analysis. Please click here to view a larger version of this figure.
Figure 2: DNAzymes targeting snR13- and snR47-dependent methylation sites in 25S rRNA. (A, B) Browser screenshots showing snR13-dependent (A) and snR47-dependent (B) methylation sites in 25S rRNA (C) 25S rRNA sequence surrounding snR13-dependent methylation site (A2281) and 10-23 DNAzyme (shown in purple) designed to cleave RNA between A2281 and U2282. A2281 is not paired with the DNAzyme while U2282 forms a pair with the first nucleotide from the DNAzyme active sequence (marked by a blue line). A blue lightning bolt marks the cleavage. (D) 25S rRNA sequence surrounding snR47-dependent methylation site (A2220) and 8-17 DNAzyme (shown in purple) designed to cleave RNA between A2220 and G2221. A2220 is not hybridized with the DNAzyme while G2221 is imperfectly paired with thymine (denoted with a dashed line). A blue lightning bolt marks the cleavage site. Please click here to view a larger version of this figure.
Figure 3: Analysis of site-specific 2’-O-methylation of 25S rRNA using 10-23 and 8-17 DNAzyme-dependent assay. (A) Analysis of snR13-dependent 25S rRNA methylation using 10-23 DNAzyme. (B) Analysis of snR47-dependent 25S rRNA methylation using 8-17 DNAzyme. RNA was visualized staining in a denaturing agarose gel. Cleavage products A and B are marked by red arrows. WT = wild-type strain; GAL = galactose, GLC = glucose. Please click here to view a larger version of this figure.
DNAzyme-dependent digestion can be used as a simple and quick method to analyze site-specific RNA 2’-O-methylation12,13. DNAzymes cleave RNA if the nucleotide upstream of the cleavage site is not methylated. In contrast to other approaches, including RNase H-directed digestion, alkaline degradation or reverse transcription in low nucleotides concentration followed by quantitative PCR or sequencing8,10,11,16, DNAzyme approach requires a simple DNA oligonucleotide and basic reagents that are present in any molecular biology laboratory. Moreover, DNAzymes may be used in a similar way to analyze RNA pseudouridylation mediated by box H/ACA snoRNA12, which makes them versatile tools in studying snoRNA targets.
DNAzyme-dependent approaches are limited only by cleavage site consensus sequences17. 10-23 DNAzymes can be used to analyze 2’-O-methylation only at position R of the RY dinucleotide, while 8-17 DNAzymes recognize the modification of the nucleotide located upstream of guanine. As a result, modifications like 2’-O-methylation of the first nucleotide in the dinucleotides guanine-adenine (GA), adenine-adenine (AA), pyrimidine-adenine (YA) and pyrimidine-pyrimidine (YY) cannot be analyzed. Moreover, the low efficiency of DNAzyme-dependent cleavage12 should be considered. Although some DNAzymes cleave RNA almost completely (Figure 3B), many DNAzymes only partially digest their targets (Figure 3B). The efficiency may depend on the sequence surrounding the cleavage site. For example, RNA regions with stretches of the same nucleotide may affect the correct positioning of the DNAzyme active sequence. Furthermore, RNA regions forming strong secondary structure may re-hybridize and suppress DNAzyme binding to the target sequence. To overcome these issues, cycles of heating and cooling of the 10-23 DNAzyme and its RNA substrate can be applied18.
We used the DNAzyme approach to investigate 2’-O-methylation of rRNA. One can also use this technique to analyze other RNA modifications, such as N6-methyladenosine19. Ribosomal RNA, due to its abundance, can be analyzed by electrophoresis and the cleavage products can be visualized under the UV light. However, this is not applicable for less abundant RNAs like RNA Polymerase II-generated coding RNAs (mRNA) and non-coding RNAs (ncRNA). These RNAs cannot usually be detected directly by RNA staining in agarose or polyacrylamide gels. In such cases, DNAzyme-dependent cleavage can be visualized by Northern blotting, indirectly detected by PCR/quantitative PCR or analyzed by quantitative PCR with polymerases (e.g., KlenTaq DNA Polymerase) capable of discriminating 2′-O-methylated RNA from unmethylated RNA20,21.
The authors have nothing to disclose.
We thank Maya Wilson and Aneika Leney for the critical reading of the manuscript. This work was supported by a Sir Henry Dale Fellowship jointly funded by the Wellcome Trust and the Royal Society (200473/Z/16/Z).
Chemicals | |||
Acid phenol | SIGMA | P4682 | |
Agarose | VWR | A2114 | |
Ammonium acetate | SIGMA | A1542 | |
Chlorophorm | Fisher scientific | 10293850 | |
DNase/RNase free water | Fischer Scientific | 10526945 | |
DNAzyme | Integrated DNA Technology | Custom oligo DNA | |
EDTA | SIGMA | E9884 | |
Ethanol Absolute | Fisher scientific | 10437341 | |
Formaldehyde | Sigma | F8775 | |
Formamide | sigma | F9037 | |
Galactose | SIGMA | G0750 | |
Gel Loading Dye | Thermo Fisher Scientific | R0611 | |
Glucose | SIGMA | G7021 | |
Glycogen | Thermo Fisher Scientific | R0561 | |
HEPES | SIGMA | H3375 | |
Isoamyl | SIGMA | W205702 | |
KCl | SIGMA | P9333 | |
MgCl2 | SIGMA | M8266 | |
MnCl2 | SIGMA | 244589 | |
MOPS | SIGMA | M1254 | |
NaCl | SIGMA | S7653 | |
Oxoid Peptone Bacteriological | Thermo Fisher Scientific | LP0037 | |
Oxoid Yeast Extract Powder | Thermo Fisher Scientific | LP0021 | |
RiboLock RNase Inhibitor (40 U/µL) | Thermo Fisher Scientific | EO0382 | |
SDS | SIGMA | 74255 | |
Sodium acetate trihydrate | SIGMA | S8625 | |
SYBR Safe DNA Gel Stain | Thermo Fisher Scientific | S33102 | |
Tris base | SIGMA | TRIS-RO | |
Name | Company | Catalog Number | コメント |
Equipment | |||
1.5 mL microtubes | Sarstedt | ||
152VR5C01M -80°C freezer | Thermo Fisher Scientific | ||
250 mL Erlenmeyer flasks | Cole-Parmer | ||
50 mL conical tubes | Sarstedt | ||
Combicup VX200 vortex | Appleton Woods | ||
DS-11 microspectrophotometer | Denovix | ||
Electrophoresis chamber (20 cm tray) | SIGMA | ||
FiveEasy F20 pH meter | Appleton Woods | ||
Gel documentation system | Syngene | ||
Heraeus Fresco 21 micro centrifuge | Fisher Scientific | ||
Megafuge 8R centrifuge with rotator suitable for 50 mL conical tubes | Fisher Scientific | ||
Mini Fuge Plus mini centrifuge | Starlab | ||
Mixer HC thermal block | Starlab | ||
OLS26 Shaking Water Bath | Grant | ||
PowerPac power supplier | BioRad |