Fig 2: m6A reader proteins YTHDF2 and YTHDF3 regulate mRNA localization to neurites. (A) Schematic representation of mRNA tethering experiments. Hippocampal neurons were transduced with a lentivirus expressing either Dendra2-5xBoxB or Dendra2-6xMS2 and with a lentivirus expressing NLS-HA-stdMCP-stdEGFP or YTHDF1/2/3-?N-HA. Sequential immunostaining for the HA tag and smFISH for Dendra2 was done at DIV7. (B) Tethering of YTHDF proteins to a reporter mRNA increases mRNA localization to neurites. Representative images for cells expressing Dendra2-6xMS2 or Dendra2-5xBoxB reporter mRNA together with YTHDF1/2/3-?N-HA proteins or stdMCP-EGFP-HA (control). Dendra2 smFISH signal is shown in red; anti-HA immunofluorescence is shown in purple. Neuron outline is shown in white. Scale bars, 5 µm. (C) Boxplots of the quantification of reporter mRNA localization as measured by smFISH. The identity of the reporter (6xMS2 = Dendra2-6xMS2; 5xBoxB = Dendra2-5xBoxB) and the protein co-expressed with the reporter are indicated. Individual values are plotted as gray dots. (D) Western blot shows knockdown of YTHDF proteins in cultured hippocampal neurons after 7 days of expression of the indicated shRNA. (E) Representative images of endogenous Map2 (upper panels) and Camk2a (lower panels) smFISH in hippocampal neurons expressing the indicated shRNAs. Neuron outline is shown in white. Scale bars, 5 µm. (F) Boxplots of the quantification of the percentage of Map2 and Camk2a mRNAs in the neurites of hippocampal neurons after expression of the indicated shRNAs. A two-way Wilcoxon-test corrected for multiple comparison was used to test significance: *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. See also Supplementary Figure S6.

Fig 3: Binding by DF proteins does not preferentially partition mRNAs to polysomes. (A) DF1 and DF2 are associated with polysomes. Representative OD260 signal trace during polysome fractionation of cells expressing DF-ADAR proteins is shown at the top. Western blot for individual endogenous DF proteins is shown below. Western blots for PABP and ribosomal proteins RPS3 and RPL10 are also shown as validation of polysome fractionation. (B) Proportion of the target mRNAs of each DF protein that are detected (FPKM ≥ 1) in polysome-associated RNA fractions. (C) Proportion of the target mRNAs of each DF protein that are edited in the polysome-associated fraction. (D) Cumulative distribution plots and box plots of the A2I editing levels in input and polysome-associated mRNAs for each DF-ADAR cell line. P-value is the result of a two-sided Wilcoxon rank-sum test. (E) Scatter plot of the cumulative RNA editing scores in input and polysome samples for each DF-ADAR cell line. mRNAs with statistically significant increased or decreased editing levels in polysome-associated RNA fractions are colored in blue and orange, respectively. The statistical significance was determined using a Wald's test (FDR < 0.05). The Pearson correlation coefficient is indicated in the top left corner. The numbers of mRNAs with increased or decreased editing are indicated in the top left and bottom right corners, respectively.

Fig 4: YTHDF2 cooperating with METTL3 regulates m6A methylation of primiR-150.a Immunofluorescence assay of YTHDF2 expression in spinal cord tissues from SNI rats at day 14 after model establishment, and red signals showed the positive staining sites. Scale bar = 100 µm. b RIP assay showed an increased binding of primiR-150 by YTHDF2, GAPDH mRNA was set as a nontarget control. ***P < 0.001. c RNA pulldown assay proved that the YTHDF2 protein was directly associated with primiR-150, EGFP RNA was used as RNA control. d RIP assay of the enrichment of primiR-150 by YTHDF2 in SNI rats overexpressed METTL3, and an increased enrichment caused by METTL3 was obtained, GAPDH mRNA was used as a nontarget control. **P < 0.01. e RNA-pulldown assay was performed in SNI rat overexpressed METTL3, and the YTHDF2 pull down by primiR-150 was significantly elevated upon upregulation of METTL3, EGFP RNA was set as RNA control. f, g Verification of the manipulations of YTHDF2 in SNI or sham rats at transcript (f) and protein levels (g). N = 3, **P < 0.01, ***P < 0.001 in comparison with the control group. h PWT of rats in the respective groups was performed when METTL3 and YTHDF2 were co-expressed. The results proved that YTHDF2 could effectively reverse the effects on PWT caused by METTL3. N = 5 biological replicates. **P < 0.01 compared with the control group. i Mature miR-150 expression was detected via qRT-PCR in sham and SNI rats after injection of METTL3 and YTHDF2. YTHDF2 could effectively reverse the effects on miR-150 expression mediated by METTL3. N = 5 biological replicates. **P < 0.01. j YTHDF2 and mature miR-150 levels were detected in 45 clinical NP patients and the two transcripts were positively expressed according to Spearman correlation test.

Fig 5: YTHDF2 recognizes the m6A site on Myh7 mRNA to promote its degradation. A Western blotting analysis of MYH7 expression in the primary cardiomyocytes transfected with si-nc or si-YTHDF2 for 24 h, then treated with 50 µg/ml CHX for the indicated times. B RT-PCR analysis of Myh7 mRNA expression in the primary cardiomyocytes transfected with si-NC or si-YTHDF2 for 24 h, then treated with ActD treatment (10 µg/ml) for the indicated times. C Amount of Myh7 mRNA in various polysome fractions was analyzed by RT-PCR. D RIP analysis of the m6A modification on the indicated regions of Myh7 mRNA (5’UTR, 5’Untranslated Region; 3’UTR, 3’Untranslated Region; CDS, Coding sequences). E Schematic diagram of the potential m6A modification sites on Myh7 mRNA. F RIP analysis of the m6A modification on the wild type (WT) or mutant (Mut) Myh7 mRNA CDS-6 region, which was cloned into pGL3-luciferase reporter plasmid. G Cells were co-transfected with pCMV3-C-FLAG-YTHDF2 (YTHDF2), pCMV3-C-FLAG-YTHDF2 mutant (YTH del), or pCMV3-C-FLAG vector (Flag), and pGL3-luciferase-wild type (WT) Myh7 mRNA CDS-6 region, pGL3-luciferase-mutant (Mut) Myh7 mRNA CDS-6 region, or pGL3-luciferase vector (Vector). RIPs were performed to detect the association between YTHDF2 or YTH-del and WT or Mut Myh7 mRNA CDS-6 region. H RNA pulldown analysis of the interaction between biotinylated-Myh7 mRNA CDS-6 (WT) or mutant Myh7 mRNA CDS-6 (Mut) and pCMV3-C-FLAG-YTHDF2 (DF2) or pCMV3-C-FLAG-YTHDF2 mutant (del). *P < 0.05