N6-methyladenosine (m6A) recruits and repels proteins to regulate mRNA homeostasis

Abstract

RNA modifications are integral to the regulation of RNA metabolism. One abundant mRNA modification is N6-methyladenosine (m6A), which affects various aspects of RNA metabolism, including splicing, translation and degradation. Current knowledge about the proteins recruited to m6A to carry out these molecular processes is still limited. Here we describe comprehensive and systematic mass-spectrometry-based screening of m6A interactors in various cell types and sequence contexts. Among the main findings, we identified G3BP1 as a protein that is repelled by m6A and positively regulates mRNA stability in an m6A-regulated manner. Furthermore, we identified FMR1 as a sequence-context-dependent m6A reader, thus revealing a connection between an mRNA modification and an autism spectrum disorder. Collectively, our data represent a rich resource and shed further light on the complex interplay among m6A, m6A interactors and mRNA homeostasis.

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Figure 1: A global m6A interactome.
Figure 2: G3BP1 and G3BP2 interact with thousands of transcripts.
Figure 3: G3BP1 protects target mRNAs from degradation.
Figure 4: FMR1 preferentially binds to m6A-containing mRNA in vitro and in vivo and affects the translation of its targets.

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Acknowledgements

We thank members of the Vermeulen lab for fruitful discussions. Work in the Vermeulen lab is supported by the NWO Gravitation program Cancer Genomics Netherlands. Work in the He lab is supported by NHGRI, NIH (HG008688). C.H. is an Investigator of the Howard Hughes Medical Institute. Work in the Carell lab is supported by Deutsche Forschungsgemeinschaft (grants SFB749, SFB1032 and SPP1784) and Bundesministerium für Bildung und Forschung (EXC114).

Author information

R.R.E. and M.V. conceived the project and wrote the manuscript, with input from all other authors. R.R.E. performed most wet-lab experiments. S.G. and M.R. prepared the m6A probes. R.G.H.L. analyzed RNA-seq and whole cell proteome data. H.S. and P.J.H. performed CLIP and PAR-CLIP experiments. Z.L. analyzed PAR-CLIP data. S.-Y.W. performed bioinformatics analysis of FMR1 PAR-CLIP data. M.P.A.B. provided technical support. P.W.T.C.J. measured mass spectrometry samples. M.M. and H.G.S. provided scientific input. C.H., T.C. and M.V. supervised the project.

Correspondence to Chuan He or Thomas Carell or Michiel Vermeulen.

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The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 A global m6A interactome.

(a) Schematic representation of the workflow used in this study (see detailed description of the approach and analysis in Online Methods). (b) Structure of the biotin molecule and linker used in the m6A probes. The RNA strand is attached to the phosphate group that is shown. (c) Mass spectra showing control and m6A probes. Calculated and observed molecular weights of the probes are indicated. The observed single peaks illustrate the purity of the probes. (d) Agarose gel showing depletion of biotin tagged probes after incubation with streptavidin beads. (e-f) Scatter plots of SILAC-based m6A pull-downs in mouse ESC nuclear extract I and mouse ESC cytoplasmic extract (f). (g-h) Scatter plots of di-methyl based m6A pull-downs in mNPC nuclear extract (g) and mNPC cytoplasmic extract (h). (i) Scatter plots of m6A pull-downs in mouse 3T3 nuclear lysates. (j-k) Scatter plots of SILAC based m6A pull downs in mESC whole cell extract using degenerate sequence probe 1 (j) and degenerate sequence probe 2 (k) The sequence context of the probes is depicted at the bottom. (l) Protein domain distribution of m6A repelled proteins in humans. (related to Fig. 1).

Supplementary Figure 2 G3BP1 and G3BP2 PAR-CLIP analysis.

(a) Scatter plot of a SILAC-based m6A pull down in mESC cytoplasmic extract in a GAm6ACU sequence context. Note the presence of G3BP1 in the background cloud. (b) The summary of PAR-CLIP sequencing reads (c) The genomic distribution of G3BP1 and G3BP2 binding sites on mRNA. (d) Enriched Most GO-terms for G3BP2 target genes. (e-f) Distribution of G3BP1 (e) and G3BP2 (f) peaks relative to YTHDF2. (g-h) Distribution of G3BP1 (g) and G3BP2 (h) peaks relative to YTHDC1 across the transcriptome. (related to Fig. 2).

Supplementary Figure 3 RNA-seq validation.

(a) Correlation between YTHDF2 mRNA binding and mRNA stability after the effect of m6A on mRNA stability was regressed out (see Online Methods). (b) Q-PCR validation showing the effect of G3BP1 KD on mRNA stability (Data are shown as range; n = 2 independent biological experiments). (c) Q-PCR validation showing the effect of G3BP1 OE on mRNA stability (Data are shown as means ± data range; n = 2 independent experiments) (related to Fig. 3).

Supplementary Figure 4 In vitro and in vivo data regarding FMR1 and m6A.

(a) FMR1 domain structure and overview of different GST-tagged FMR1 deletion constructs (b) GST western blot analysis of GST-FMR1 deletion constructs binding to unmodified and m6A-containing RNA probes (GGACU context). For all constructs, the correct size band is indicated with a red arrow. (c) Western blots showing expression of FLAG-HA, FLAG-HA-FMR1 and FLAG-HA-FMR1 I304N mutant (FMR1-m) in HEK293 cells. (d) Representative LC/MS based quantification showing levels of m6A in FLAG-HA-FMR1 and FLAG-HA-FMR1 mutant enriched fraction, prior to RiboMinus treatment. Data are shown as range; n = 2 independent biological experiments. (e) Venn diagram showing the overlap between FMR1 (mouse brain) and YTHDF1 (HEK cells) target transcripts. (f) GO-term enrichment analysis of common target transcripts of FMR1 (mouse brain) and YTHDF1 (HEK cells). GO terms related to neurogenesis are indicated in red. (g) Box-plots depicting the global changes in protein half-lives in the indicated samples. (related to Fig. 4).

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–4 (PDF 1022 kb)

Life Sciences Reporting Summary (PDF 159 kb)

Supplementary Table 1

RNA probes and oligos (XLSX 12 kb)

Supplementary Data Set 1

Methods and uncropped images. (PDF 8355 kb)

Supplementary Data Set 2

Mass spectrometry data from RNA pulldowns. (XLSX 6909 kb)

Supplementary Data Set 3

G3BP1 and G3BP2 PAR-CLIP data. (XLSX 3279 kb)

Supplementary Data Set 4

mRNA half-life estimation RNA-seq data. (XLSX 623 kb)

Supplementary Data Set 5

Pulse-SILAC data. (XLSX 725 kb)

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Edupuganti, R., Geiger, S., Lindeboom, R. et al. N6-methyladenosine (m6A) recruits and repels proteins to regulate mRNA homeostasis. Nat Struct Mol Biol 24, 870–878 (2017). https://doi.org/10.1038/nsmb.3462

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