Methylation
Uncovering the mechanisms of gene regulation using microarrays
A review of Detection and discovery of RNA modifications using microarrays.
Note: This is a review of the published article listed below. All information, quotes, figures, methods, and findings mentioned in this review are from that article, and are the property of its authors and/or the publication in which the article originally appeared.
Hiley and colleagues (2005) examined, in detail, whether any of the 17 different types of RNA covalent modifications could be detected using oligonucleotide microarrays. The group designed custom high-density oligonucleotide microarrays through Agilent Technologies, using probes that were complementary to known non-coding RNA sequences and flanking regions, and were tiled at 5 nt intervals for most RNAs (intron-containing mRNAs were tiled every 20 nt and mitochondrial RNAs every 15 nt). The use of microarray technology enabled the researchers to detect hundreds of known modification events, as well as several novel modification sites. A tRNA modification involving the Gcd10/Gcd14 complex was verified by subsequent primer extension analysis. The success of this interrogation of the genome is the first example of using microarray as a genome-wide tool for RNA modification detection and sets the stage for extending the scope of this type of analysis.
Table 1
Known and predicted yeast non-coding RNAs included on the microarray
| ncRNA | Number of transcripts | Tiling frequency |
|---|
|
| 35S pre rRNA | 1 | 5 |
| 5S rRNA | 1 | 5 |
| Genomic tRNAs | 70 | 5 |
| snoRNAs | 84 | 5 |
| snRNAs | 6 | 5 |
| RNase P | 1 | 5 |
| RNase MRP | 1 | 5 |
| SRP RNA | 1 | 5 |
| Telomerase RNA | 1 | 5 |
| RUFs | 8 | 5 |
| Introns | 236 | 20 |
| Spliced junctions | 236 | 5 |
| Mitochondrial genome features | 44 | 15 |
| mRNA 3′ ends | 8 | 20 |
Figure 1. Detection of covalent modification by microarray.
(A) Modification disrupts base pairing between RNA and probe. Wild-type tRNA LysCTT (top) contains a dimethylguanosine residue at position 26, which disrupts pairing with the probe. trm1-Δ tRNA LysCTT (bottom) lacks this modification and can pair completely with the probe (see also Figure 2B). A schematic diagram of the tRNA is shown below. Rectangles represent probes complementary to tRNA sequence, and thin lines represent probes complementary to 5’ and 3’ genomic flanking regions. The relative fluorescence of each probe is indicated by color-coded rectangles above the schematic diagram (according to the scale on the right); the tRNA nucleotides covered by each oligo are shown. (B) Analysis of strains defective for tRNA modification. tRNA oligos (ordered from 5’ to 3’) versus individual experiments (described below the figure) are plotted. Oligos to which there was significantly better binding in the mutant tRNA samples are indicated by red color, as shown by the color-bar in (A). Groups of probes covering tRNA nucleotides modified by each enzyme are outlined in blue rectangles. The type of modification and positions known to be modified by each enzyme are shown. Only tRNA probes with ratios at least 2-fold above wild type are shown.
Figure 2. tRNA methylation analyzed by microarray.
(A) Three different types of detectable methylation. Unique tRNA probes with ratios of at least 2 are color coded according to the scale shown and displayed from 5’ to 3’ of the tRNA sequence. The tRNA isoforms and specific nucleotides covered are shown to the right of the figure. Oligos predicted to be affected in the each experiment are outlined with blue rectangles. (B) Schematic representation of selected tRNAs. One tRNA from each of the experiments in which the methylation defect was detected is shown in schematic form as described in Figure 1A. Functional groups involved in Watson–Crick base pairing are circled in blue; modifications are circled in red.
Figure 3. Novel modification events.
(A) Potential new targets for Trm5 and the Gcd10/Gcd14 complex. Modifications and target sites are proposed for three tRNAs whose RNA sequences have not been published and RNA modification profiles are unknown. (B) Demonstration of m1A58 modification in tRNAGlnCUG. Inferred RNA sequence of tRNAGlnCUG showing the position of the primer used to detect m1A modification at position 58 (highlighted in blue). The four positions that are underlined are the residues of this minor tRNA species that differ from the sequence of the other two previously characterized tRNAGln isoforms (both tRNAGlnUUG). The residues found at those positions in tRNAGlnUUG are shown in parentheses above. (C) Primer extension analysis of RNA derived from either gcd14-Δ (lane 1) or wild-type cells (lane 2). Lanes C, T, A and G are sequencing lanes of the primer extended RNA. (D) Two elongation-specific tRNA Met oligos show formamide-dependent differential hybridization in GCD10/GCD14 mutants. A schematic diagram and the corresponding values in the chart show that probes covering Mete nucleotides 11–25 and 16–30 exhibited high ratios in both experiments targeting the GCD10–GCD14 complex. tRNA and probe sequences are shown below; the overlapping region, Mete 16-25, is outlined in red. The table to the right shows the difference in ratio of representative probes for two formamide concentrations.
Title: Detection and discovery of RNA modifications using microarrays.
Journal: Nucleic Acids Res. 2005 Jan 7;33(1):e2.
Authors: Hiley SL, Jackman J, Babak T, Trochesset M, Morris QD, Phizicky E, Hughes TR.
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