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. 2017 Oct;24(10):870-878.
doi: 10.1038/nsmb.3462. Epub 2017 Sep 4.

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

Raghu R Edupuganti  1 , Simon Geiger  2 , Rik G H Lindeboom  1 , Hailing Shi  3 , Phillip J Hsu  3 , Zhike Lu  3 , Shuang-Yin Wang  1 , Marijke P A Baltissen  1 , Pascal W T C Jansen  1 , Martin Rossa  2 , Markus Müller  2 , Hendrik G Stunnenberg  1 , Chuan He  3 , Thomas Carell  2 , Michiel Vermeulen  1
Affiliations

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

Raghu R Edupuganti et al. Nat Struct Mol Biol. 2017 Oct.

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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Conflict of interest statement

COMPETING FINANCIAL INTERESTS

The authors declare no competing financial interests.

Figures

Figure 1
Figure 1
A global m6A interactome. (a,b) Results of a SILAC-based m6A RNA pulldown in HeLa nuclear extract (a) and cytoplasmic lysates (b). m6A-interacting proteins are depicted in blue, repelled proteins are in orange, and background proteins are in black. We used a threshold of 1.5× the interquartile range to identify significant interactors. In the box plots, center lines indicate medians, box edges represent the interquartile range (IQR), and whiskers extend to ±1.5× the IQR; outlier values are shown as black circles. (c,d) GO-term enrichment analysis (Online Methods) of m6A readers (c) and repelled proteins (d) in HeLa cells. The relationships between various GO terms are depicted by gray bars. The intensity of the red shading indicates the significance of the relationship (darker shading denotes greater significance). (e) The overlap between m6A readers identified in mESCs and differentiated cells (NIH 3T3). (f) The overlap between m6A-repelled proteins in mESCs and NIH 3T3 cells. The Venn diagrams summarize data obtained in three independent experiments. (g) Protein domains present in m6A interactors in mouse (top) and human (bottom) cells. (h) Many m6A interactors have also been identified in previously published in vivo mRNA-protein interaction studies–. Source data for g and h are available online. Proteomics data and GO enrichment analysis are presented in Supplementary Data Set 2.
Figure 2
Figure 2
G3BP1 and G3BP2 interact with thousands of transcripts. (a) The G3BP1 domain structure. (b) GST western blot showing RNA-sequence-context-dependent inhibition of GST-G3BP1 binding to RNA by m6A. Molecular weights are indicated on the right. (c,d) RNA cross-link immunoprecipitation in HEK293 cells expressing either Flag-G3BP1 or Flag-G3BP2. Flag western blots (c) and radioactively labeled RNA-protein complexes (d) are shown. OE, overexpression; Ctrl, control. (e) Common peaks between PAR-CLIP biological replicates 1 and 2 for G3BP1 (top left) and G3BP2 (top right), and comparisons between G3BP1 and G3BP2 peaks (bottom left) and target genes (bottom right). (f) Metagene profiles of G3BP1 and G3BP2 distribution across the transcriptome. (g) The most enriched consensus sequences of G3BP1 (top) and G3BP2 (bottom) on mRNA; P values were determined as described in the Online Methods. (h) The most significantly enriched GO terms among G3BP1 target genes. The numbers to the right of the bars represent the number of genes in that GO term. (i) The number of G3BP1 and G3BP2 peaks that overlap with m6A peaks among m6A-containing mRNAs. (j) The number of G3BP1 and G3BP2 peaks on m6A-containing mRNAs that overlap with m6A. (k) The distribution of G3BP1 (left) and G3BP2 (right) relative to m6A sites at single-nucleotide resolution. The distance between PAR-CLIP peaks and m6A sites was counted in 10-nucleotide bins. A Gaussian kernel smoothing over the histogram is plotted as a transparent black line. The uncropped blot image for b is shown in Supplementary Data Set 1. Source data for i and j are available online. G3BP1 and G3BP2 PAR-CLIP data and related GO term enrichment analysis are presented in Supplementary Data Set 3.
Figure 3
Figure 3
G3BP1 protects target mRNAs from degradation. (a) The correlation between the number of m6A peaks on mRNA molecules and mRNA half-life. (b) The correlation between YTHDF2 mRNA binding and mRNA stability. (c) The correlation between G3BP1 mRNA binding and target gene transcript stability. In a–c, n represents the number of genes, center lines in box plots indicate medians, box edges represent the interquartile range, and whiskers extend to ±1.5× the interquartile range; outlier values are not shown. (d) mRNA half-life measurements after G3BP1 knockdown (KD) or overexpression (OE). ActD, actinomycin D. (e) qPCR analysis of G3BP1 levels after protein knockdown or overexpression. GAPDH was used to normalize expression. Data are shown as the mean ± s.e.m.; n = 3 independent experiments. (f) Western blots depicting knockdown and overexpression of G3BP1. Ponceau-stained protein bands are shown as loading controls. Con, control plasmid expressing Flag tag. (g) Cumulative distribution function plots showing the effect of G3BP1 knockdown on target genes. The log2 fold changes in mRNA half-life were grouped and analyzed on the basis of the number of G3BP1-binding sites on each transcript. P values were calculated by two-sided Mann–Whitney U test. (h) Western blots depicting knockdown of METTL3 and G3BP1. Ponceau-stained protein bands are shown as loading controls. (i) A cumulative distribution function plot showing the effect of METTL3 knockdown on G3BP1 target genes. (j) A cumulative distribution function plot showing the effect of combined G3BP1 and METTL3 knockdown on G3BP1 target genes. log2 fold changes were grouped and analyzed as described for g. Uncropped blot images for f and h are shown in Supplementary Data Set 1. A summary of mRNA half-lives as determined by RNA-seq analysis is presented in Supplementary Data Set 4.
Figure 4
Figure 4
FMR1 preferentially binds to m6A-containing mRNA in vitro and in vivo and affects the translation of its targets. (a) Western blots showing preferential m6A binding by recombinant GST-FMR1 in vitro. (b) Quantification of FMR1 binding to control and m6A probes (data are shown as mean ± s.e.m.; n = 3 independent experiments). (c) Predominant consensus sequences for m6A, and consensus mRNA-binding sites for FMR1 isoforms 1 and 7 (ref. 48). (d) Visualization of binding of FMR1 isoforms 1 and 7 to mRNA relative to known m6A sites on mRNA,. Randomized peaks were generated from the transcriptome and used as a control as described in the Online Methods. (e) Immunofluorescence analysis of the expression of Flag-HA (FH)-tagged FMR1 isoform 1 and Flag-HA-tagged FMR1 isoform 1 I304N mutant (mut) in HEK293T cells. (f) RNA cross-linking and immunoprecipitation in lysates from HEK293 cells expressing either Flag-HA-tagged FMR1 or Flag-HA-tagged FMR1-I304N, using anti-Flag. IP, immunoprecipitate; FT, flow-through; M, molecular weight ladder. (g) Representative LC-MS quantification showing enrichment of m6A in Flag-HA-tagged FMR1–bound mRNA. Error bars represent the range; n = 2 independent experiments. (h) The overlap between FMR1 (ref. 48) and YTHDF1 target transcripts. (i) A schematic representation of the pulsed SILAC workflow. Dox, doxycycline. (j) Western blots showing the results of METTL3 knockdown and inducible FMR1/FMR1 mutant protein expression in HeLa cells. Ponceau-stained protein bands are shown as loading controls. (k) Representative peptide spectra showing SILAC label incorporation for a peptide belonging to a protein whose translation rate is not affected by FMR1 overexpression (left) and a peptide belonging to a protein that is translationally repressed by FMR1 overexpression (right) (compare the incorporation of medium-heavy (M) and heavy (H) label in the two plots). (l) A cumulative distribution function plot showing the effect of METTL3 knockdown (siMETTL3), FMR1 overexpression, and FMR1-I304N overexpression on translation rates in HeLa cells. We used the time until half the protein was labeled as a proxy for the translation rate (Online Methods). A summary of the proteomics data from the pulsed SILAC analyses is provided in Supplementary Data Set 5. Uncropped blot images for a, f and j are shown in Supplementary Data Set 1. Source data for g are available online.
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References

    1. Dimock K, Stoltzfus CM. Sequence specificity of internal methylation in B77 avian sarcoma virus RNA subunits. Biochemistry. 1977;16:471–478. - PubMed
    1. Desrosiers RC, Friderici KH, Rottman FM. Characterization of Novikoff hepatoma mRNA methylation and heterogeneity in the methylated 5′ terminus. Biochemistry. 1975;14:4367–4374. - PubMed
    1. Zheng G, et al. ALKBH5 is a mammalian RNA demethylase that impacts RNA metabolism and mouse fertility. Mol Cell. 2013;49:18–29. - PMC - PubMed
    1. Jia G, et al. N6-methyladenosine in nuclear RNA is a major substrate of the obesity-associated FTO. Nat Chem Biol. 2011;7:885–887. - PMC - PubMed
    1. Liu J, et al. A METTL3-METTL14 complex mediates mammalian nuclear RNA N6-adenosine methylation. Nat Chem Biol. 2014;10:93–95. - PMC - PubMed