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1 SUPPLEMENTARY INFORMATION doi: /nature11112 Figure 1 Salient features of the m 6 A methylome. a, m 6 A punctuates RNA molecules mainly around the stop codon and in unusually long internal exons. Methylation is catalyzed by a nuclear macromolecular complex, with only one known subunit (METTL3). Demethylation is carried out by FTO. The methylation consensus motif RRACU is an orderr of magnitude more prevalent that the observed frequency of m 6 A. b, Methylation is enriched in multi-isoform genes and correlates with differentially spliced sequences, arguingg in favor of a role for m 6 A in splicing control. Moreover, m 6 A modulates the interaction with RNA-binding proteins. c., m 6 A methylation is highly conserved between human and mouse, with respect to both global methylome features (consensus and methylation profile along gene architecture) and specific positions in many orthologous genes. Yellow hexagon, m 6 A; thick bars, exons; thin bars, introns; vertical lines, methylation consensus motif. 1

2 RESEARCH SUPPLEMENTARY INFORMATION Figure 2 Examples of m 6 A peaks in the indicated gene transcripts. a-c, coding genes; d, non-coding genes. Coverage of m 6 A (IP) and control (input) fragments is indicated in blue and red, respectively. Significant peaks (above threshold) are highlighted in yellow. Black dashed lines signify CDS borders; transcript architecture is shown beneath, with thin parts corresponding to UTRs and thicker ones to CDS; exon- exon junctions are indicated by vertical black lines. 2

3 SUPPLEMENTARY INFORMATION RESEARCH Figure 3 Assessment of the mean number of m 6 A peaks per 1000 nts. Analysis was performed using peaks within genes that are expressed over a set quantile threshold. For example, within genes expressed in the top 10% (quantile threshold = 0.9), 0.45 sites are detected per 1000 nts. The number of reported peaks most probably underestimate the actual number due to the conservative thresholds applied, sequence coverage (leading to saturation in peak detection only for highly expressed genes) and the nature of the dataset containing a single, intron-free splice variant per gene. 3

4 RESEARCH SUPPLEMENTARY INFORMATION a b c Figure 4 Consensus motifs for m 6 A methylation. a, Volcano plot depicting the motifs enriched within m 6 A fragments. Each point represents a 4-6 nt long motif. Every motif is assigned a fold-change and a P-valuee based on comparison of its prevalence in m 6 A fragments with control fragments. Motifs are colored by their similarity to the previously determined m 6 A consensus motif. b, c, De-novo peaks (b) and MACS- motif finder MEME identified similar motifs in transcriptome-identified identified peaks (c). Both motifs contain the core motif. Each analysis was performed on the top-scored 1,000 peaks. Of note, de-novo MEME analysis of the 1,000 top-scored MACS-identified peaks identified a longer, less degenerate motif, WGRACW (W= A or U). The consensus is far more prevalent than the actual observed frequency of m 6 As by an order of magnitude - a total of 9,778 peaks were detected in 8,041 adequately expressed genes (>40 reads/kb), as compared to 99,777 methylation consensus sitess in the same genes. The height of a nucleotidee at a given position reflects its frequency. 4

5 SUPPLEMENTARY INFORMATION RESEARCH Figure 5 Correlations of m 6 A peaks with genomic positions. The number of enriched windows distanced up to 1000 nts from a set of various genomic or randomly selected positions is shown. Results are shown also for the mock windows serving as additional negative controls. The X axis denotes the distance between the center of the window and one of the following 6 positions: CDS startt and end sites, a randomly selected position from the gene (random), the center of the gene (center), the transcription start and end sites (TSS and trans. end, respectively). 5

6 RESEARCH SUPPLEMENTARY INFORMATION Figure 6 Enrichment of m 6 A peaks in long exons. For each length threshold (up to 2000 nts) we assessed the enrichment of m 6 A peaks within internal exons exceeding that threshold. Enrichment was calculated by dividing the number of m 6 A peaks present within internal exons by the proportion of the transcriptome (excluding terminal exons) covered by them. a b Figure 7 Characteristics of TSS peaks. a, Chart depicting the fraction of genes beginning with each of the four nucleotides, as a functionn of the distance between the first detectedd m 6 A or negative control peaks from TSS. Distances are presented in intervals of 200 nts, up to 1000 nts. Error bars represent the standard error of the mean (SEM). b, Box plots depicting the distribution of distances between the detected peaks and the nearest consensus methylation sequence according to genee segment. 6

7 SUPPLEMENTARY INFORMATION RESEARCH Figure 8 Support for the non-monotonic relationship between m 6 A peaks and gene expression levels. Reads weree subsampled in each gene so as not to exceed a threshold of 50 reads/kb, corresponding to lower percentiles of the data. The resulting distribution of peaks across the different gene segments as a function of gene expression depicts a trend similar to the one observed based on the entire data (Fig. 2d), suggesting that our result is not a bias of differential read sampling. 7

8 RESEARCH SUPPLEMENTARY INFORMATION a b c d Figure 9 Analyses of the mouse liver methylome. a, Volcano plot depicting the motifs enriched within m 6 A fragments. Each point represents a 4-6 nt long motif. Every motif is assigned a fold-change and a P-value based on comparison of its prevalence in m 6 A fragments with control fragments. Motifs are colored according to their similarity with the previously determined m 6 A consensus motif. b, Density plot showing the distribution of m 6 A/control peaks according to exon length. c, Box plots depicting the distribution of distances between the detected peaks and the nearest consensus methylation sequence in each gene segment. As in humans, m 6 A peaks in the TSS segment do not appear near a methylation consensus sequence. d, Chart depicting the fraction of genes beginning with each of the four nucleotides, as a function of the distance between the first detected m 6 A or negative control peaks and TSS. Distances are presented in intervals of 200 nts, up to 1000 nts. Error bars represent the standard error of the mean (SEM). m 6 A peaks in the TSS are strongly correlated with presence of adenosines at the first position of the transcript, suggesting they may be attributable to cap methylation. 8

9 SUPPLEMENTARY INFORMATION RESEARCH Figure 10 Human-mouse orthologous genes with conserved m 6 A peaks. Gene names are indicated. Coverage of m 6 A (IP) and control (input) fragments is indicated in blue and red, respectively. Significant peaks (above threshold) are highlighted in yellow. Black dashed lines signify CDS borders; transcript architecture is shown beneath, with thin parts corresponding to UTRs and thicker ones to CDS; exon- exon junctions are indicated by vertical black lines. 9

10 RESEARCH SUPPLEMENTARY INFORMATION Figure 111 Comparison of m 6 A peak positions across experimental conditions and tissues. a, Heat maps depicting the proportion of shared m 6 A peak positions between any two experiments. Each map represents m 6 A peaks in the category indicated above it. Values equal the proportion of m 6 A peaks common to X and Y out of all m 6 A peaks in X, and vice versa. A peak is considered present in Y if it exists within 50 nts of its position in X. Note that the heat map is not symmetric: the proportion of sites in Y which are present in X does not necessarily equal the proportion of sites in X present in Y. Peaks are generally more conserved in the stop codon segment than in other segments; in protein-coding genes than in non-coding ones; and in long internal exons compared to their shorter counterparts. Lower degrees of conservationn were evident in categories containing a small fraction of m 6 A peaks (right panels), hence bearing a small impact on the overall high conservation. b, An unchanged methylation profile across different growing conditions and cell types is demonstrated. UT, untreated; UV, ultraviolet; IFN, interferon γ; HGF, hepatocyte growth factor; HS, heat shock. 10

11 SUPPLEMENTARY INFORMATION RESEARCH Figure 12 METTL3 KD in HepG2 cells. METTL3 was knocked down for 7 days. KD was validated by a, Western blot analysis b, real-time subjected to TUNEL assay quantitative RT-PCRrevealing high levels of apoptosis of KD cells compared to control After 7 days of METTL3 silencing, c,, Cells weree cells. Figure 13 Differential expression analysis of METTL3 KD and control cells. a, Scatter plot of log2 ratio (FC) versus mean expression. Genes that are differentially expressed are indicated in different colors according to their associated m 6 A peaks (black- genes with no peaks; yellow- genes with exon-associated and exon-associated peaks). Genes peaks; red- genes with peaks in introns; cyan- genes with both intronexhibiting insignificant differential expression are presented in gray. Horizontal lines mark the FC significance. Vertical line marks moderate expression level. Overall, more down-regulated genes were observed. Genes with peak-containing introns were significantly down-regulated. b, Up- and down-regulationn of differentially expressed genes. Down-regulated genes are enriched with m 6 A peaks. * indicates slight significance (P < 0.05). **-indicates high significance (P < 0.01). 11

12 RESEARCH SUPPLEMENTARY INFORMATION Figure 14 m 6 A peaks are overrepresented in alternatively spliced exons. m 6 A peak summitss were assigned to 10 categories of spliced exons according to Ensembl coding genes. a, The distribution of m 6 A-containing exons (red) across the different categories of splicing events compared to the exon distribution in the dataset (blue). b, Distribution of m 6 A-containing exons among constitutive and non-constitutive splicing categories. P values are indicated above each category. Alt., alternative; Const., constitutive; ME, mutually exclusive. 12

13 SUPPLEMENTARY INFORMATION RESEARCH Figure 15 METTL3 KD in HepG2 cells affects the p53 signaling pathway. a, Differentially expressed genes and splice variants are significantly enriched with p53 signaling pathway members. Genes exhibiting differential expression at the genee level are colored red; genes exhibiting alternative splicing differences and differential splice variant expression are colored blue. b, MDMX splice variants. The isoforms that show reduced expression are indicated in arrows. Other isoforms were not significantly changed. Changed isoforms include the p53 binding domain 13

14 RESEARCH SUPPLEMENTARY INFORMATION Figure 16 Characterization of the effects of yeast IME4 deletion: Gene ontology analysis of differentially expressed genes under meiosis-inducing conditions, in ime4/ime44 compared to its parental strain was performed using DAVID tool Yeast cells have a single homologue of METTL3,, called IME4. Inactivation of IME4 was shown to result in the loss of m 6 A in mrna of mutant cells 1. As expected, undetectable RNA amounts were obtained when RNA isolated from the ime4 mutant strain was subjected to IP using our m 6 A-seq protocol. m 6 A-seq of RNA isolated from wt (SK1 strain) cells grown in both vegetative and meiosis-inducing media was performed. Although sufficient RNA amounts and reads were obtained (in 2 independent experiments), no significant peaks could be identified, suggesting that the inducedd level of methylation in sporulating yeast is below the sensitivity of our protocol. We have analyzed the effects of IME4 deletion on vegetative and meiotic cells. Expression microarray analyses of mutant and wt strains, 4 hrs after transfer to meiosis-inducing media, as welll as under vegetative growth conditions, were performed. Vegetatively growing ime4 cells showed increased expression of the RME1 gene (repressor of meiosis) which prevents precocious entry into the meiotic program 2. Surprisingly, despite the fact that a diploid strain was analyzed, there was also a striking change in the expression level of haploid-specific genes (MAPK pathway), suggesting that RNA methylation may be used to enforce the sexual identity of diploid cells, required for the correct enforcement of the gametogenesis program. Consistently, when cells were induced to undergo meiosis, ime4 diploids failed to undergo the meiotic divisions. The mutants showed a reducedd level of expression of genes involved in ribosome biology (rrna processing, ribosomal and nucleolar proteins), as well as reduced expression of IME1 and IME2, the two known inducers of meiosis 2. Thus, the yeast IME4 genee plays an important role in the regulation of the developmental switch from vegetative cells into gametogenesis. 14

15 SUPPLEMENTARY INFORMATION RESEARCH Figure 17 m 6 A peaks in various IRES elements. Coverage of m 6 A (IP) and control (input) fragments is indicated in blue and red, respectively. Significant peaks (above threshold) are highlighted in yellow. Black dashed lines signify CDS borders; transcript architecture is shown beneath, with thin parts corresponding to UTRs and thicker ones to CDS; exon-exon junctions are indicated by vertical black lines. 15

16 RESEARCH SUPPLEMENTARY INFORMATION Figure 18 RNA affinity chromatography identifies putative m 6 A- binding proteins. a, Schematic of the RNA affinity chromatography approach using a bait corresponding to a part of the RSV genome. Colored shapes are illustrative of bound proteins; m, m 6 A. b, Scatter plot of proteins bound to methylated versus unmethylated (control) RNA baits. Enriched proteins (Methods) were assigned red, green and yellow dots according to the number of experiments in which they were detected 3 out of 3, 2 out of 3 and 1 out 3, respectively. Proteins that did not pass the enrichment threshold are represented by blue dots. While the methylated bait retrieved several significantly enriched proteins, the unmethylated control bait did not, demonstrating the specificity of our approach. c, Overlap between YTH binding consensus (highlighted yellow) and methylation consensus (red letters) superimposed on the sequence of the RNA bait. Asterisk represents m 6 A. d, Western blot validation of significant proteins. 16

17 SUPPLEMENTARY INFORMATION RESEARCH Reference to Figures 1 Bodi, Z., Button, J. D., Grierson, D. & Fray, R. G. Yeast targets for mrna methylation. Nucleic Acids Res 38, (2010). 2 Kassir, Y. et al. Transcriptional regulation of meiosis in budding yeast. International review of cytology 224, (2003). Links 2HighScoringPeaks.html link 1 Additional visualized examples representing the top 100 gene profiles harboring the highest-scoring peaks. 17

18 RESEARCH SUPPLEMENTARY INFORMATION Tables Sample Total identified peaks coding genes noncoding genes % peaks in TSS segment % peaks in 5' UTR segment % peaks in CDS segment % peaks in stop codon segment % peaks in 3' UTR segment % internal peaks in long exons HepG2-UT % 2.8% 36.6% 27.6% 20.5% 86.6% HepG2-UV % 2.9% 39.0% 27.2% 21.4% 82.0% HepG2-IFN % 1.6% 47.8% 29.0% 20.4% 70.3% HepG2-HGF % 1.8% 44.4% 29.4% 20.5% 82.5% HepG2-HS % 2.8% 40.8% 27.4% 20.6% 85.9% Human Brain % 1.7% 47.5% 28.3% 20.3% 87.4% Mouse Liver % 1.2% 29.5% 39.2% 24.8% 91.1% Table 1 Summary of m 6 A peaks in various experiments and their distribution. UT, untreated; UV, ultraviolet; IFN, interferon γ; HGF, hepatocyte growth factor; HS, heat shock; TSS, transcription start site; UTR, untranslated region; CDS, coding sequence; HepG2, human hepatocellular carcinoma cell line. Sample # Reads # unique reads (genome) # reads mapped to transcriptome % reads in top 20 genes HumanHepG2UT_Input HumanHepG2UT_IP HumanHepG2UV_Input HumanHepG2UV_IP HumanHepG2IFN_Input HumanHepG2IFN_IP HumanHepG2HGF_Input HumanHepG2HGF_IP HumanHepG2HS_Input HumanHepG2HS_IP HumanBrain_Input HumanBrain_IP MouseLiver_Input MouseLiver_IP Table 2 Alignment statistics. UT, untreated; UV, ultraviolet; IFN, interferon γ; HGF, hepatocyte growth factor; HS, heat shock; HepG2, human hepatocellular carcinoma cell line; IP, immunoprecipitation. 18

19 SUPPLEMENTARY INFORMATION RESEARCH # Peaks # Genes Total 20,401 - Annotated Ensembl genes 18,713 10,355 Coding 18,423 9,846 Non-coding Intron (only) 1,508 1,073 Table 3 MACS-identified peaks within Ensembl annotated genes (hg18). To allow a broader view on peak distribution within introns and exons absent from the reference transcriptome, reads were re-aligned to the human genome and peaks were identified with MACS peak calling algorithm. 20,401 peaks were identified (FDR 5%, fold-change (FC) 4), containing over 96% of the transcriptome-identified peaks above. Most of the peaks (18,423/20,401) reside in protein coding genes. 1,508 peaks localize in introns. This number probably underestimates the actual number of methylated introns due to their relatively low abundance in poly(a) + -enriched samples and to exclusion of peaks falling into transcript-dependent intron/exon sequences. Peaks were considered as intronic only if their summit did not overlap any exon sequence. Category Differentially expressed Differentially expressed and m 6 A methylated Genes 1,977 1,218 Transcript isoform 7,251 4,636 Exon intron 2, Table 4 Differential expression analysis of METTL3 KD. Analysis parameters were set to FC 2, FDR 5%. 19

20 RESEARCH SUPPLEMENTARY INFORMATION Gene Symbol Total # of peptides with m 6 A bait Total # of peptides with control bait ELAVL YTHDF YTHDF DBN DHX HNRNPA2B GNAI HNRNPR 24 0 FUBP PTBP DSP 59 0 HNMPH SYNCRIP 44 0 PRPF SFPQ 25 0 POLR2E 21 0 MCM PPP1CA out of 3 experiments 2 out of 3 experiments 1 out of 3 experiments Table 5 Mass-spectrometry results 20

21 SUPPLEMENTARY INFORMATION RESEARCH Notes 1. Measures taken to ensure validity and stringency of m 6 A-seq: a) Elution of anti-m 6 A antibody-bound RNA fragments was only achieved when we used an excess of free m 6 A nucleosides. b) In contrast to IP from mrna of sporulating WT yeast, m 6 A-deficient mrna, obtained from an ime4 mutant (ortholog of METTL3), yielded undetectable RNA amounts. c) High signal reproducibility was demonstrated in several independent biological experiments. d) Identification of a known m 6 A site within 18S rrna was confirmed in all experiments (FC = 5). e) Of note, the anti- m 6 A antibody was raised against the modified nucleoside, and therefore introduces no sequence bias. 2. Comparison of the human brain and HepG2 methylomes: although the transcriptome of normal human brain contains a set of brain-specific genes that are not expressed in HepG2, a significant fraction of the peaks identified in the brain (591 peaks, 66%) overlapped with those identified in HepG2 ( Fig. 11b). The remaining brain peaks all localize to neuronalspecific genes. legends to supplementary Tables 6-8 Table 6 Dataset of all identified m 6 A peaks in HepG2 cell line and normal human brain id KnownCanonical identification chr chromosome txstart transcription start position on chromosome txend Transcription end position on chromosome strand - +/- chromosome strand genesymbol official symbol of gene desc description Factor_iscoding either coding or noncoding, depending on the annotation of the gene in the UCSC table. Meanexpquant expression quantile of the gene based on reads in input experiment Peaksnum number of peaks identified in gene Peakspos position of peaks relative to beginning of transcript (after removing introns) Peaksexonnum ordinal number of exons in which the peaks were identified Peaksexlengths lengths of the exons in which the peaks were found Peakscores the peakscore (see Methods) of the identified peaks Annotpeakpos segment along the gene (see Manuscript) in which the peaks Table 7 Dataset of all identified m 6 A peaks in mouse liver. Legend as in data

22 RESEARCH SUPPLEMENTARY INFORMATION Table 8 Differential m 6 A peaks between various experimental conditions Peakspos position of peaks relative to beginning of transcript (after removing introns) Peakscores the peakscore (see Methods) of the identified peaks Peaksexonnum ordinal number of exons in which the peaks were identified Peaksexlengths lengths of the exons in which the peaks were found factor_relpeakpos Transcript segment in which peak appears exp - denotes the two compared experiments, where the first one denotes the one in which a peaks was present, and the second - the one in which a peak was absent. Thus, HumanHEPG2HeatShockvsHumanHEPG2HGF accompanies a peak that was present under heat shock conditions and absent under HGF treatment. 22