. 2012 Jun 22;149(7):1635-46.
Epub 2012 May 17.
Comprehensive Analysis of mRNA Methylation Reveals Enrichment in 3' UTRs and Near Stop Codons
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Comprehensive Analysis of mRNA Methylation Reveals Enrichment in 3' UTRs and Near Stop Codons
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Methylation of the N(6) position of adenosine (m(6)A) is a posttranscriptional modification of RNA with poorly understood prevalence and physiological relevance. The recent discovery that FTO, an obesity risk gene, encodes an m(6)A demethylase implicates m(6)A as an important regulator of physiological processes. Here, we present a method for transcriptome-wide m(6)A localization, which combines m(6)A-specific methylated RNA immunoprecipitation with next-generation sequencing (MeRIP-Seq). We use this method to identify mRNAs of 7,676 mammalian genes that contain m(6)A, indicating that m(6)A is a common base modification of mRNA. The m(6)A modification exhibits tissue-specific regulation and is markedly increased throughout brain development. We find that m(6)A sites are enriched near stop codons and in 3' UTRs, and we uncover an association between m(6)A residues and microRNA-binding sites within 3' UTRs. These findings provide a resource for identifying transcripts that are substrates for adenosine methylation and reveal insights into the epigenetic regulation of the mammalian transcriptome.
Copyright © 2012 Elsevier Inc. All rights reserved.
Figure 1. Specificity and Sensitivity of m
A. Dot blot analysis demonstrates antibody specificity for m 6A. Increasing amounts of an oligonucleotide containing either m 6A or unmodified adenosine were spotted onto a membrane and probed with the m 6A antibody. While increased m 6A immunoreactivity is observed in the presence of increasing concentrations of the m 6A oligonucleotide (top), only background levels of immunoreactivity are observed at the highest concentrations of the A oligonucleotide (bottom). Blots shown are representative of results from three experiments. B. Competition dot blot assays were performed on membranes spotted with 100 ng of m 6A-containing oligonucleotide. Antibody binding to the m 6A oligonucleotide is attenuated by pre-incubation with increasing amounts of m 6A-containing competitor RNA (top), but not with RNA containing unmodified adenosine (bottom). Amount of competitor RNA used (left to right): 0 ng (0 nM), 10 ng (0.1 nM), 100 ng (1.1 nM), 1 µg (11.2 nM). Blots shown are representative of results from four experiments. C. Competition dot blot assays were performed as in (B). Antibody was pre-incubated with increasing amounts of N 6-methyladenosine triphosphate ( N 6-MeATP), adenosine triphosphate (ATP), N 1-methyladenosine triphosphate ( N 1-MeATP), or 2’- O-methyladenosine triphosphate (2’- O-MeATP). Only N 6-MeATP is able to compete with antibody binding. Concentration of competitor nucleotide used (left to right): 0 µM, 1 µM, 2 µM, 4 µM. Blots shown are representative of results from three experiments. D. Detection of m 6A in cellular DNA. Genomic DNA isolated from dam+ (containing m 6A) or dam− (lacking m 6A) E. coli was sheared and subjected to immunoblotting with the anti-m 6A antibody. Although 1.5 times as much DNA from dam− E. coli was loaded (left panel), the antibody only recognizes the m 6A present in DNA from dam+ E. coli (right panel). Blot shown is representative of results from three experiments. See also Figure S2.
Figure 2. Distribution and Dynamic Cellular Regulation of m
6A in RNA
A. Widespread distribution of m 6A levels in a variety of tissues. Total RNA isolated from mouse brain, heart, lung, liver, and kidney (top) was subjected to m 6A immunoblot analysis. Ethidium bromide staining of the 28S rRNA is shown as a loading control (bottom). B. Quantification of m 6A abundance within various tissues. Quantification of m 6A immunoreactivity in (A) was measured by densitometry and normalized to the intensity of the corresponding 28S rRNA band for each tissue ( n = 3; data are presented as mean ± SEM). C. m 6A is enriched within mRNAs. Oligo(dT) Dynabeads were used to isolate poly(A) RNA from total mouse brain RNA, and the unbound “flow-through” RNA was saved as the poly(A)-depleted fraction. Equal amounts of total RNA, poly(A) RNA, and poly(A)-depleted RNA were then subjected to m 6A immunoblot analysis (top). Ethidium bromide staining of 28S rRNA is shown as a loading control (bottom). Intense m 6A immunoreactivity is observed in the poly(A) RNA fraction, consistent with high levels of m 6A within mRNAs. D. Depletion of poly(A) tails from mRNA does not reduce levels of m 6A in mRNA. Poly(A) RNA was isolated from total mouse brain RNA using oligo(dT) Dynabeads. Half the sample was then subjected to poly(A) tail depletion by hybridizing to oligo(dT) primers and digestion with RNase H. Immunoblot analysis with the m 6A antibody (top panel) shows that levels of m 6A in poly(A) RNA (left) and poly(A) tail-depleted RNA (right) are comparable. Removal of poly(A) tails was confirmed using 3’RACE and RTPCR to detect β-actin; no product is detected in the tail-depleted sample when oligo(dT) primers are used for cDNA synthesis (middle panel). As a control, use of random hexamers successfully generates a product in both samples (bottom panel). See also Figure S1.
Figure 3. Regulation of m
6A Levels in Cells and During Development
A. Ontogeny of m 6A abundance throughout brain development. Total RNA was isolated from mouse brain at embryonic day 18 (E18), postnatal day 0 (P0), postnatal day 14 (P14), and adulthood, then subjected to immunoblot analysis to detect m 6A-containing transcripts. Ethidium bromide staining of 28S rRNA bands is shown as a loading control. B. FTO demethylates a wide range of cellular transcripts. FTO was expressed in HEK293T cells for 48h, and cellular RNA was subjected to immunoblot analysis to detect m 6A. See also Figure S1.
Figure 4. Outline of MeRIP-Seq Protocol and Distribution of Sequencing Reads
A. Schematic representation of MeRIP-Seq. Total RNA is subjected to RiboMinus treatment to remove rRNA species. RNAs containing m 6A are then immunoprecipitated by mixing the RNA with m 6A antibody-coupled Dynabeads. m 6A-containing RNAs are then eluted from the antibody-coupled beads and subjected to a second round of m 6A immunoprecipitation. The resulting RNA pool, which is highly enriched for m 6A-containing RNAs, is then subjected to next-generation sequencing. B. Schematic of sequencing reads and their alignment to locations in the genome surrounding an m 6A site. Top: an mRNA that contains a single m 6A residue along its length. Middle: individual 100 nt-wide mRNA fragments which are isolated following m 6A immunoprecipitation, each of which contains the same m 6A residue from the mRNA depicted above. Bottom: histogram showing predicted frequency of MeRIP-Seq reads obtained by sequencing individual immunoprecipitated fragments. Read frequency is predicted to increase with closer proximity to the m 6A site, forming a “peak” which is roughly 200 nt wide at its base and 100 nt wide at its midpoint. C. Sequencing reads from MeRIP-Seq converge over m 6A sites. Representative UCSC Genome Browser plot from MeRIP-Seq data which demonstrates typical read frequency peak formation surrounding a site of m 6A (shown here is the 3’ UTR of Pax6). Peak height is displayed as reads per base per million mapped reads (BPM). See also Figures S2, S3.
Figure 5. Validation of m
6A Targets and Characteristics of m 6A Localization
A. Different sequencing platforms and antibodies result in similar m 6A profiles. UCSC Genome Browser tracks displaying read clusters from three MeRIP-Seq replicates (MeRIP1, MeRIP2, and MeRIP3) are shown along the length of the Ldlr transcript. The upper-most track (non-IP) represents the non-immunoprecipitated control sample. B. Validation of m 6A-containing mRNA identified with MeRIP-Seq. Hybridization-based RNA pulldown was used to isolate Ldlr mRNA from total brain RNA, followed by confirmation of m 6A presence (arrow) by immunoblot analysis with anti-m 6A. A control sample using a non-specific probe of equal size (Control Probe) was run in parallel. Total mouse brain RNA (Input RNA) is shown as a reference for m 6A labeling. C. Transcriptome-wide distribution of m 6A peaks. Pie charts showing the percentage of m 6A peaks (top) and non-IP sample reads (bottom) within distinct RNA sequence types. m 6A is highly enriched in 3’ UTRs and CDSs compared to the distribution of reads in the non-IP samples. D. Distribution of m 6A peaks across the length of mRNA transcripts. 5’ UTRs, CDSs, and 3’ UTRs of RefSeq mRNAs were individually binned into regions spanning 1% of their total length, and the percentage of m 6A peaks that fall within each bin was determined. The moving averages of mouse brain peaks percentage (red) and HEK293T peak percentage (blue) are shown. E. Highly similar m 6A peak distribution is observed within many human and mouse transcripts. UCSC Genome Browser plots showing MeRIP-Seq read clusters in the representative transcript SREK1. MeRIP-Seq reads cluster at the same distinct regions of SREK1 in both HEK293T cell RNA (top) and mouse brain RNA (bottom). See also Figures S4 – S7, Tables S1-S6.
Figure 6. MeRIP-Seq Reveals Features of m
6A in mRNA
A. Phylogenetic conservation of m 6A peaks. PhyloP scores of m 6A peak regions were compared to those of randomly shuffled regions throughout gene exons. There was a significantly higher median conservation score (K-S test, * p ≤ 2.2e −16) in m 6A peaks (0.578) than in the random regions (0.023). B. Sequence motifs identified within m 6A peaks. The motif G[AG]ACU and variants thereof ([AC]GAC[GU], GGAC, [AU][CG]G[AG]AC, and UGAC) was highly enriched in m 6A peaks. Additionally, one U-rich motif (bottom right) was identified as being significantly underrepresented within m 6A peaks. Color bars under each motif indicate the degree of underrepresentation (blue) or overrepresentation (yellow) within regions of m 6A peaks in the non-IP control sample (CNTL) and the MeRIP sample (MeRIP). C. m 6A motif sequences frequently lie near the center of m 6A peaks. Shown is a plot of the cumulative distribution of m 6A motif positions within m 6A peaks containing a single motif. Motifs cluster in the center of peaks, suggesting that the methylated adenosines in these motifs account for the m 6A peaks identified in MeRIP-Seq. D. Example of a m 6A motif sequence near the center of a peak. UCSC Genome Browser plot containing tracks for MeRIP-Seq reads (red) and non-IP control reads (black) at the Ilf2 locus. The m 6A peak within the Ilf2 3’ UTR contains a single m 6A motif identified in ( B). The sequence of this motif (highlighted in yellow) is located at the center of the m 6A peak. E. Distribution of m 6A peaks and miRNA target sites within 3’ UTRs. The frequency of m 6A peaks (blue) and miRNA target sites (red) along the length of 3’ UTRs is shown. F. Association between 3’ UTR methylation and miRNA abundance. The 25 most abundant miRNAs in brain have a significantly greater percentage of m 6A peaks within their target mRNA 3’ UTRs than do the 25 most weakly expressed brain miRNAs (*p<0.05, Wilcoxon test). The error bars in A and F indicate the highest and lowest values, and the box boundaries denote the 1st quartile, median, and 3rd quartile. See also Figures S3, S6, and S7.
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