Research ArticleMOLECULAR BIOLOGY

Single-base mapping of m6A by an antibody-independent method

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Science Advances  03 Jul 2019:
Vol. 5, no. 7, eaax0250
DOI: 10.1126/sciadv.aax0250
  • Fig. 1 m6A identification method based on m6A sensitive RNA endoribonuclease.

    (A) Validation for the methylation sensitivity of endoribonuclease MazF by synthetic m6A-containing RNA oligonucleotide. (B) Validation for the methylation sensitivity of endoribonuclease ChpBK. (C) Various ratios of m6A-containing oligo mixed with unmethylated oligo digested by endoribonuclease. The m6A/A ratios of synthetic oligo are indicated to imitate practical RNAs with different m6A ratios. (D) Relative grayscale of proportionally mixed RNA oligo digested by endoribonuclease. (E) The schematic diagram of m6A-REF-seq.

  • Fig. 2 Transcriptome-wide distribution of m6A revealed by m6A-REF-seq.

    (A) The analysis pipeline of m6A-REF-seq data. (B) The snapshot of sequencing reads shows a known m6A site in 18S rRNA. (C) Overlap sites among three replicates after removing RNA secondary structure. (D) The overall shift of methylation ratio after FTO treatment. The value of methylation change indicates the methylation ratio of each m6A site in MazF minus that after FTO treatment. (E) Transcriptome-wide distribution of m6A. Pie chart shows the percentages of m6A sites located within CDS, 5′UTR, and 3′UTR. (F) Single-base m6A sites from m6A-REF-seq show a typical transcriptome-wide distribution pattern of m6A. (G) Overlap of m6A sites to the m6A peaks identified by antibody-based method. (H) The proportion of motifs containing m6A sites. The red square includes the RRACA motif and the green square includes the DRACA motif.

  • Fig. 3 Single-base validation using ligation-amplification method and qPCR.

    (A) Schematic diagram of ligation-amplification method for single-base m6A validation. (B) Validation results of six individual sites. Five of the sites are validated to be m6A sites, whereas the other one is confirmed to be not modified. Data for m6A-CLIP, miCLIP-CITS, miCLIP-CIMS, and MeRIP-seq are downloaded from published literatures. (C) Schematic diagram of qPCR to quantify the methylation level of a specific m6A site. The undigested mRNA sample is treated as control. (D) qPCR results and methylation ratios of six m6A sites. H1 to H3 represent the highly methylated sites (>0.75), while L1 to L3 represent the weakly methylated sites (<0.35). The left y axis represents the ΔΔCt values, and the right y axis represents the methylation ratio determined by m6A-REF-seq. GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

  • Fig. 4 Single-base method reveals high resolution features of m6A.

    (A) The distance of individual m6A sites to the stop codons. DRACA and DRACH background motifs are extracted from the transcriptome. (B) The relative position of m6A sites within exons. (C) The length distribution of m6A-containing exons versus all exons in m6A-modified genes. (D) The distances between two m6A sites are significantly enriched within 200 bp regions compared to random sampling (Fisher’s exact test, **P < 0.01).

  • Fig. 5 Conservation of m6A in mammals.

    (A) Metagene plots of m6A in the brain of human, mouse, and rat. (B) Shared m6A-modified genes among three species. (C) Diagram showing the m6A sites conserved in the corresponding short regions from different species. (D) Frequency of distances for pairwise m6A in brain. Randomly picked ACA motifs are assigned for the same analysis as control. (E) Conservation scores of all m6A sites, methylation sites in ortholog genes, and conserved m6A sites are compared to that for all A sites in ACA motifs (P < 9 × 10−10, Wilcoxon test).

Supplementary Materials

  • Supplementary material for this article is available at http://advances.sciencemag.org/cgi/content/full/5/7/eaax0250/DC1

    Fig. S1. Quantitative demonstration of various fractions of m6A-containing oligo mixed with unmethylated oligo digested by ChpBK.

    Fig. S2. FTO demethylate assay.

    Fig. S3. The base composition of mRNA products cleaved by MazF.

    Fig. S4. The single-site validation for 18S rRNA control site and m6A site.

    Fig. S5. The single-site validation of eight m6A sites and one unmethylated site.

    Fig. S6. Quantitative PCR results of six m6A sites.

    Fig. S7. Metagene plots of m6A in mouse heart and mouse testis.

    Fig. S8. Conservation of m6A in mammalian kidney.

    Fig. S9. Conservation of m6A in mammalian liver.

    Fig. S10. Gene expression and the expected number of shared m6A-modified genes.

    Fig. S11. Evolutionary conservation between methylated and unmethylated A sites in the same genes of brain tissue.

    Fig. S12. Evolutionary conservation between methylated and unmethylated A sites in the same genes of kidney tissue.

    Fig. S13. Evolutionary conservation between methylated and unmethylated A sites in the same genes of liver tissue.

    Fig. S14. Conservation scores for brain.

    Fig. S15. Validation for the methylation sensitivity of mutated MazF-K56A.

    Table S1. Basic information of sequencing data for human HEK293T cell line.

    Table S2. Designed probes and universal primers for T3 ligase–based validation.

    Table S3. Designed primers for Quantitative PCR validation.

    Table S4. Basic information of sequencing data for mammalian tissues.

    Table S5. m6A sites identified by m6A-REF-seq for mammalian tissues.

    Table S6. Shared m6A-modified genes and m6A sites between pairwise species and the significance.

  • Supplementary Materials

    This PDF file includes:

    • Fig. S1. Quantitative demonstration of various fractions of m6A-containing oligo mixed with unmethylated oligo digested by ChpBK.
    • Fig. S2. FTO demethylate assay.
    • Fig. S3. The base composition of mRNA products cleaved by MazF.
    • Fig. S4. The single-site validation for 18S rRNA control site and m6A site.
    • Fig. S5. The single-site validation of eight m6A sites and one unmethylated site.
    • Fig. S6. Quantitative PCR results of six m6A sites.
    • Fig. S7. Metagene plots of m6A in mouse heart and mouse testis.
    • Fig. S8. Conservation of m6A in mammalian kidney.
    • Fig. S9. Conservation of m6A in mammalian liver.
    • Fig. S10. Gene expression and the expected number of shared m6A-modified genes.
    • Fig. S11. Evolutionary conservation between methylated and unmethylated A sites in the same genes of brain tissue.
    • Fig. S12. Evolutionary conservation between methylated and unmethylated A sites in the same genes of kidney tissue.
    • Fig. S13. Evolutionary conservation between methylated and unmethylated A sites in the same genes of liver tissue.
    • Fig. S14. Conservation scores for brain.
    • Fig. S15. Validation for the methylation sensitivity of mutated MazF-K56A.
    • Table S1. Basic information of sequencing data for human HEK293T cell line.
    • Table S2. Designed probes and universal primers for T3 ligase–based validation.
    • Table S3. Designed primers for Quantitative PCR validation.
    • Table S4. Basic information of sequencing data for mammalian tissues.
    • Table S5. m6A sites identified by m6A-REF-seq for mammalian tissues.
    • Table S6. Shared m6A-modified genes and m6A sites between pairwise species and the significance.

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