Comprehensive analysis of mRNA methylation reveals enrichment in 3' UTRs and near stop codons

Abstract

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.

Figure 1. Specificity and Sensitivity of m⁶A-Specific Antibody

A. Dot blot analysis demonstrates antibody specificity for m⁶A. Increasing amounts of an oligonucleotide containing either m⁶A or unmodified adenosine were spotted onto a membrane and probed with the m⁶A antibody. While increased m⁶A immunoreactivity is observed in the presence of increasing concentrations of the m⁶A 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 m⁶A-containing oligonucleotide. Antibody binding to the m⁶A oligonucleotide is attenuated by pre-incubation with increasing amounts of m⁶A-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 N⁶-methyladenosine triphosphate (N⁶-MeATP), adenosine triphosphate (ATP), N¹-methyladenosine triphosphate (N¹-MeATP), or 2’-O-methyladenosine triphosphate (2’-O-MeATP). Only N⁶-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 m⁶A in cellular DNA. Genomic DNA isolated from dam+ (containing m⁶A) or dam− (lacking m⁶A) E. coli was sheared and subjected to immunoblotting with the anti-m⁶A antibody. Although 1.5 times as much DNA from dam− E. coli was loaded (left panel), the antibody only recognizes the m⁶A 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 m⁶A in RNA

A. Widespread distribution of m⁶A levels in a variety of tissues. Total RNA isolated from mouse brain, heart, lung, liver, and kidney (top) was subjected to m⁶A immunoblot analysis. Ethidium bromide staining of the 28S rRNA is shown as a loading control (bottom). B. Quantification of m⁶A abundance within various tissues. Quantification of m⁶A 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. m⁶A 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 m⁶A immunoblot analysis (top). Ethidium bromide staining of 28S rRNA is shown as a loading control (bottom). Intense m⁶A immunoreactivity is observed in the poly(A) RNA fraction, consistent with high levels of m⁶A within mRNAs. D. Depletion of poly(A) tails from mRNA does not reduce levels of m⁶A 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 m⁶A antibody (top panel) shows that levels of m⁶A 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 m⁶A Levels in Cells and During Development

A. Ontogeny of m⁶A 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 m⁶A-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 m⁶A. 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 m⁶A are then immunoprecipitated by mixing the RNA with m⁶A antibody-coupled Dynabeads. m⁶A-containing RNAs are then eluted from the antibody-coupled beads and subjected to a second round of m⁶A immunoprecipitation. The resulting RNA pool, which is highly enriched for m⁶A-containing RNAs, is then subjected to next-generation sequencing. B. Schematic of sequencing reads and their alignment to locations in the genome surrounding an m⁶A site. Top: an mRNA that contains a single m⁶A residue along its length. Middle: individual 100 nt-wide mRNA fragments which are isolated following m⁶A immunoprecipitation, each of which contains the same m⁶A 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 m⁶A 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 m⁶A sites. Representative UCSC Genome Browser plot from MeRIP-Seq data which demonstrates typical read frequency peak formation surrounding a site of m⁶A (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 m⁶A Targets and Characteristics of m⁶A Localization

A. Different sequencing platforms and antibodies result in similar m⁶A 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 m⁶A-containing mRNA identified with MeRIP-Seq. Hybridization-based RNA pulldown was used to isolate Ldlr mRNA from total brain RNA, followed by confirmation of m⁶A presence (arrow) by immunoblot analysis with anti-m⁶A. 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 m⁶A labeling. C. Transcriptome-wide distribution of m⁶A peaks. Pie charts showing the percentage of m⁶A peaks (top) and non-IP sample reads (bottom) within distinct RNA sequence types. m⁶A is highly enriched in 3’ UTRs and CDSs compared to the distribution of reads in the non-IP samples. D. Distribution of m⁶A 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 m⁶A 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 m⁶A 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 m⁶A in mRNA

A. Phylogenetic conservation of m⁶A peaks. PhyloP scores of m⁶A 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 m⁶A peaks (0.578) than in the random regions (0.023). B. Sequence motifs identified within m⁶A peaks. The motif G[AG]ACU and variants thereof ([AC]GAC[GU], GGAC, [AU][CG]G[AG]AC, and UGAC) was highly enriched in m⁶A peaks. Additionally, one U-rich motif (bottom right) was identified as being significantly underrepresented within m⁶A peaks. Color bars under each motif indicate the degree of underrepresentation (blue) or overrepresentation (yellow) within regions of m⁶A peaks in the non-IP control sample (CNTL) and the MeRIP sample (MeRIP). C. m⁶A motif sequences frequently lie near the center of m⁶A peaks. Shown is a plot of the cumulative distribution of m⁶A motif positions within m⁶A peaks containing a single motif. Motifs cluster in the center of peaks, suggesting that the methylated adenosines in these motifs account for the m⁶A peaks identified in MeRIP-Seq. D. Example of a m⁶A 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 m⁶A peak within the Ilf2 3’ UTR contains a single m⁶A motif identified in (B). The sequence of this motif (highlighted in yellow) is located at the center of the m⁶A peak. E. Distribution of m⁶A peaks and miRNA target sites within 3’ UTRs. The frequency of m⁶A 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 m⁶A 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.