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.

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

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

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

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.