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. 2019 Nov 25;10(1):5332.
doi: 10.1038/s41467-019-13317-9.

m6A in mRNA coding regions promotes translation via the RNA helicase-containing YTHDC2

Yuanhui Mao  1 , Leiming Dong  1 , Xiao-Min Liu  1 , Jiayin Guo  2 , Honghui Ma  3 , Bin Shen  2 , Shu-Bing Qian  4
Affiliations

m6A in mRNA coding regions promotes translation via the RNA helicase-containing YTHDC2

Yuanhui Mao et al. Nat Commun. .

Abstract

Dynamic mRNA modification in the form of N6-methyladenosine (m6A) adds considerable richness and sophistication to gene regulation. The m6A mark is asymmetrically distributed along mature mRNAs, with approximately 35% of m6A residues located within the coding region (CDS). It has been suggested that methylation in CDS slows down translation elongation. However, neither the decoding feature of endogenous mRNAs nor the physiological significance of CDS m6A has been clearly defined. Here, we found that CDS m6A leads to ribosome pausing in a codon-specific manner. Unexpectedly, removing CDS m6A from these transcripts results in a further decrease of translation. A systemic analysis of RNA structural datasets revealed that CDS m6A positively regulates translation by resolving mRNA secondary structures. We further demonstrate that the elongation-promoting effect of CDS methylation requires the RNA helicase-containing m6A reader YTHDC2. Our findings established the physiological significance of CDS methylation and uncovered non-overlapping function of m6A reader proteins.

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Conflict of interest statement

The authors declare no competing interests.

Figures

Fig. 1
Fig. 1
CDS m6A is enriched in transcripts with inactive translation. a Distribution of m6A sites along transcripts with different regional methylation. Transcripts are grouped into 5′ UTR, CDS, and 3′ UTR methylation based on the identified m6A sites. Only METTL3 or WTAP sensitive m6A sites are considered. The distribution of total m6A sites was shown in gray. UTR: untranslated region, CDS: protein coding region. b A Venn diagram shows the overlapping of transcripts containing m6A sites in different regions. c Translation efficiency (TE, left column) and m6A density across the transcriptome are presented as parallel heat-maps. Note the inverse correlation between CDS m6A methylation and TE. d Translation efficiency (TE) is plotted as accumulative fractions for mRNAs with different amount of m6A peaks in CDS. e A representative example (MAPK) of evolutionary conserved m6A sites between MEF and HEK293 cells. The coverage of m6A is shown as black lines and the input as gray. f A box plot shows the translation efficiency (TE) of mRNAs containing conserved (C) and non-conserved (NC) m6A sites in MEF and HEK293 cells (Wilcox test, all P values < 2.2 × 10−16). The median of TE in each group is indicated by a center line, the box shows the upper and lower quantiles, whiskers shows the 1.5× interquartile range, and the outliers are indicated by points. Source data are provided as a Source Data file.
Fig. 2
Fig. 2
Characterize the role of CDS m6A in translation efficiency. a Aggregation plots show the mean ribosome densities along mRNA regions aligned to the RRAC motif with (pink line) or without (blue line) m6A modification. Right panels show the mean ribosome densities along mRNA regions aligned to GAA or GAC codons with (pink line) or without (blue line) m6A modification. A minus position value indicates upstream of m6A sites, whereas a positive value indicates downstream of m6A sites. The m6A site in RRAC motif or codons is highlighted by red. b The fold change of translation efficiency upon METTL3 knockdown is plotted as accumulative fractions (Wilcox test, P = 1.9 × 10−8) for mRNAs bearing CDS methylation (m6A+) or not (m6A−). Both groups have similar levels of basal TE. c Aggregation plots show the mean ribosome densities along mRNA regions aligned to the RRAC motif with (top panel) or without (bottom panel) m6A modification. Plots from cells with or without METTL3 knockdown are color coded. The m6A site in RRAC motif is highlighted by red. d From the same data sets as c, the fold change of ribosome accumulation upstream of the motif in response to METTL3 knockdown is calculated for transcripts with or without m6A modification (Wilcox test, P < 0.001). For the boxplot in d and e, the median value in each group is indicated by a center line, the box shows the upper and lower quantiles, whiskers shows the 1.5× interquartile range, and the outliers are indicated by points. e A histogram shows the distribution of changes of regional ribosome density in response to METTL3 knockdown. A sliding window of 30 nt in length with a step of 3 nt are used to calculate the local ribosome density. Regions with <1/3-fold (Dec, blue) and >3-fold (Inc., red) changes are highlighted by color coding. The right box plot shows the predicted minimum folding free energy (MFE) for regions with <1/3-fold (Dec) and >3-fold (Inc.) changes (Wilcox test, P < 2.2 × 10−16). Source data are provided as a Source Data file.
Fig. 3
Fig. 3
CDS m6A methylation resolves mRNA secondary structures. a The predicted minimum folding free energy (MFE) is plotted along mRNA regions surrounding the CDS RRAC motif with (pink line) or without (blue line) m6A modification. A sliding window with 30 nt in length and a step of 3 nt was used to calculate MFE. For each window, the central position is used for alignment. A minus position value indicates upstream of m6A sites, whereas a positive value indicates downstream of m6A sites. The m6A site in RRAC motif is highlighted by red. Notably, a lower MFE value indicates a higher potential for RNA secondary structures. b The GC content is plotted along mRNA regions surrounding the CDS RRAC motif with (pink line) or without (blue line) m6A modification. c The in vivo icSHAPE signal is plotted along mRNA regions surrounding the CDS RRAC motif with (pink) or without (blue) m6A modification. Notably, a higher in vivo icSHAPE signal indicates a less structured region. The right boxplot shows the average of icSHAPE signals across mRNA regions from –500 nt to 500 nt relative to the RRAC motif with (pink) or without (blue) m6A modification (Wilcox test, ***P < 0.001). The median of icSHAPE signals in each group is indicated by a center line, the box shows the upper and lower quantiles, whiskers shows the 1.5× interquartile range. The outliers are not shown. d The left panel shows the schematic of a dual luciferase reporter with a sandwiched secondary structure derived from MALAT1 (2556–2587). Both UU → CC and A → G mutants are also shown. The m6A site is highlighted by red. The right panel shows the ratio of Rluc/Fluc in transfected cells expressing wild type or indicated mutants. Error bars, mean ± s.e.m.; Single-tailed t test, n = 4, *P < 0.05, **P < 0.01. e The ratio of Rluc/Fluc in transfected cells expressing wild type or mutant reporters, with either METTL3 or METTL14 knockdown. Error bars, mean ± s.e.m.; single-tailed t test, n = 4, *P < 0.05, **P < 0.01. Source data are provided as a Source Data file.
Fig. 4
Fig. 4
YTHDC2 promotes translation efficiency by acting on CDS m6A. a Puromycin labeling assay shows the global protein synthesis in HEK293 cells lacking each individual cytoplasmic m6A reader proteins. The right panel shows the quantitative results based on puromycin signals normalized with β-actin. Error bars, mean ± s.e.m.; t est, n = 3, *P < 0.05. DF1: YTHDF1, DF2: YTHDF2, DF3: YTHDF3, DC2: YTHDC2. b Distribution of the binding sites of cytoplasmic m6A reader proteins across the human transcriptome. All binding sites are identified from PAR-CLIP data sets obtained from HeLa cells. The distribution of m6A sites is shown as gray. c The fold change of translation efficiency upon YTHDC2 knockdown is plotted as accumulative fractions (Wilcox test, P < 2.2 × 10−16) for transcripts bearing CDS methylation (m6A+) or not (m6A−). Both groups have similar levels of basal TE. d A histogram shows the distribution of changes of regional ribosome density in response to YTHDC2 knockdown. A sliding window of 30 nt in length with a step of 3 nt are used to calculate the local ribosome density. Regions with <1/3-fold (Dec, blue) and >3-fold (Inc., red) changes are highlighted by color coding. The right box plot shows the predicted minimum folding free energy (MFE) for regions with <1/3-fold (Dec) and >3-fold (Inc.) changes (Wilcox test, P < 2.2 × 10−16). The median of MFE in each group is indicated by a center line, the box shows the upper and lower quantiles, whiskers shows the 1.5× interquartile range, and the outliers are indicated by points. e The ratio of Rluc/Fluc in transfected cells expressing wild type or mutant reporters, with or without YTHDC2 knockdown. Error bars, mean ± s.e.m.; Single-tailed t test, n = 4, *P < 0.05. Source data are provided as a Source Data file.
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