USP1/UAF1-Stabilized METTL3 Promotes Reactive Astrogliosis and Improves Functional Recovery after Spinal Cord Injury through m⁶A Modification of YAP1 mRNA

USP1/UAF1 complex interacts with METTL3. A, Silver-stained gel of astrocytic proteins coimmunoprecipitated using anti-Flag magnetic beads and eluted by Flag peptides. B, Mass spectrometry identified specific peptides of USP1 (top) and UAF1 (bottom) in immunoprecipitated METTL3 complexes. C–E, Heat maps of a set of deubiquitinating enzymes in GSE5296 (C), GSE42828 (D), and scratch injured astrocytes (E). F–H, Relative USP1 expression in GSE5296 (F), GSE42828 (G), and RNA sequence of scratch injured astrocytes (H); **p = 0.0011, ***p < 0.001. I–K, Relative UAF1 expression in GSE5296 (I), GSE42828 (J), and RNA sequence of scratch-injured astrocytes (K); *p = 0.0127, **p < 0.01. L, Relative mRNA expression of USP1 and UAF1 at the indicated times after SCI was examined by RT-qPCR; ***p < 0.001. M, N, Relative protein expression of USP1 and UAF1 at the indicated times after SCI were examined and quantified; ***p < 0.001. O, Relative mRNA expression of astrocytic USP1 and UAF1 at the indicated times after scratch injury was examined by RT-qPCR; ***p < 0.001. P, Q, Relative protein expression of astrocytic USP1 and UAF1 at the indicated times after scratch injury was examined and quantified; **p = 0.0025, ***p < 0.001. R, Protein extracts from primary astrocytes were subjected to immunoprecipitation with anti-METTL3 or anti-IgG and then immunoblotted with indicated antibodies. S, At 48 h after scratch injury, equal amounts of astrocytic protein lysates were immunoprecipitated using anti-METTL3 and examined by immunoblotting with indicated antibodies. T, Protein lysate of primary astrocytes was immunoprecipitated with anti-USP1 or anti-UAF1 and then evaluated by immunoblotting with anti-METTL3. U, V, Extracts from HEK 293T cells transfected with Flag-METTL3 and Myc-USP1 (U) or Myc-UAF1 (V) were immunoprecipitated with anti-Flag, followed by immunoblotting with anti-Myc and anti-Flag. W, Primary astrocytes transfected with Myc-USP1 or Myc-UAF1 were immunofluorescence stained using anti-METTL3 and anti-Myc. Scale bar, 10 µm. Correlation was analyzed using ImageJ software. X, Molecular dock of METTL3 and USP1/UAF1 complex. Y, Schematic diagram of Flag-tagged full-length METTL3 and corresponding truncated mutants. Z, HEK 293T cells were transfected with Myc-USP1 (left) or Myc-UAF1 (right) along with Flag-tagged METTL3 or its mutants. Cell lysates were immunoprecipitated with anti-Myc, followed by immunoblotting with indicated antibodies. One-way ANOVA followed by post hoc Bonferroni correction (F, G, I, J, L, N, O, Q); Student’s two-tailed unpaired t test (H, K).

USP1/UAF1 complex deubiquitinates and stabilizes METTL3. A, Expression of astrocytic METTL3 was evaluated by Western blotting following overexpression of USP1 or UAF1 with or without addition of 30 mm ML323 for 4 h; *p < 0.05, **p = 0.0054, ***p < 0.001. B. Effects of USP1 or UAF1 knockdown on astrocytic METTL3 protein expression were assessed via Western blotting; **p < 0.01, ***p < 0.001. C, D, Analysis of astrocytic METTL3 level after USP1 (C) or UAF1 (D) depletion with or without treatment of 10 µm proteasome inhibitor MG132 for 4 h; ***p < 0.001. E, Effects of USP1 or UAF1 silencing on remaining astrocytic METTL3 level at the indicated times after translation inhibition was evaluated by Western blotting; ***p < 0.001. F, G, Cell lysates of primary astrocytes transfected with Myc-USP1 and Myc-USP1 C90S mutant (F) or Myc-UAF1 (G) were immunoprecipitated with anti-METTL3, followed by immunoblotting with indicated antibodies. H, Ubiquitination levels of METTL3 in astrocytes silencing USP1 or UAF1 were determined via immunoprecipitation using anti-METTL3, followed by immunoblotting with indicated antibodies. I, J, Proteins of HEK 293T cells transfected with Flag-METTL3, HA-Ub, Myc-USP1, Myc-USP1 C90S (I), and Myc-UAF1 (J) were subjected to immunoprecipitation using anti-Flag and then immunoblotted with indicated antibodies. K, Following USP1 or UAF1 depletion, lysates from HEK 293T cells transfected with Flag-METTL3 and HA-Ub were immunoprecipitated with anti-Flag and then immunoblotted with indicated antibodies. L, M, Lysates from HEK 293T cells transfected with HA-Ub, HA-K48-Ub, HA-K63-Ub, Flag-METTL3, Myc-USP1 (L), and UAF1 (M), followed by immunoprecipitation with anti-Flag and subsequent immunoblotting with anti-HA. N, O, Coimmunoprecipitation analysis of HEK 293T cells transfected with HA-K48-Ub, HA-K48R-Ub, Flag-METTL3, Myc-USP1 (N), and Myc-UAF1 (O), followed by immunoprecipitation with anti-Flag and then immunoblotting with anti-HA. One-way ANOVA followed by post hoc Bonferroni correction (A–D); two-way ANOVA followed by post hoc Bonferroni correction (E); nonlinear regression of one phase decay (E).

Knockdown of astrocytic METTL3 inhibits astrogliosis and wound healing in vitro. A–C, The RNA methylation levels of primary astrocytes after METTL3 knockdown were evaluated using a m⁶A dot blot (A), colorimetric m6A quantification assay (B), and LC-MS/MS analysis (C); ***p < 0.001. D, GFAP protein expression of primary astrocytes transfected with shRNA-control (shNC) or shRNA-METTL3 (shMETTL3) after scratch injury. E, Representative immunofluorescence images of GFAP and EdU in primary astrocytes transfected with shNC or shMETTL3 after scratch injury. F–H, Quantification of wound healing ability (F), EdU⁺ ratio (G), and protrusion length (H) of astrocytes transfected with shNC or shMETTL3; **p = 0.0027, ***p < 0.001. Student’s two-tailed unpaired t test (B, C, F–H). Scale bar, 100 µm.

Conditional METTL3 deletion in astrocytes has no effects on spinal cord development and motor functions without SCI. A, Schematic strategy of METTL3^fl/fl mice construction. B–E, Efficiency of METTL3 deletion in cerebellum, cortex, hippocampus, spinal cord, as well as cultured astrocytes were detected by Western blots; ***p < 0.001. F, G, Representative immunofluorescence images of GFAP and METTL3 in cultured astrocytes (F) and quantification of astrocytic sizes (G). Scale bar, 50 µm. H–K, Nissl staining and immunofluorescence staining of neurons comparing spinal cords from Mettl3^fl/fl mice with Mettl3^CKO mice and quantification of neuron number. Scale bar, 100 µm. L, Body weight of Mettl3^fl/fl mice and Mettl3^CKO mice at different development stages. M, BMS score of Mettl3^fl/fl and Mettl3^CKO mice without SCI. N, O, Representative images of gait from Mettl3^fl/fl and Mettl3^CKO mice (N) and quantification of stride length and width (O). Student’s two-tailed unpaired t test (C, E, G, I, K, M, O). Two-way ANOVA followed by post hoc Bonferroni correction (L).

Conditional METTL3 deletion in astrocytes hinders functional recovery and suppresses reactive astrogliosis after SCI. A, Schematic diagram of functional analysis after SCI. B–D, RNA m⁶A methylation levels of Mettl3^fl/fl and Mettl3^CKO mice were evaluated by dot blot (B), colorimetric m⁶A quantification assay (C), and LC-MS/MS assay (D); **p = 0.0063, ***p < 0.001. E, Protein expression and quantification of GFAP in 4 mm spinal cord centered on the lesion core of Mettl3^fl/fl and Mettl3^CKO mice at the indicated times after SCI; *p < 0.05, **p = 0.0037. F, Sensory behaviors by von Frey test of Mettl3^fl/fl and Mettl3^CKO mice at baseline and day 28 postinjury; ***p < 0.001. G, BMS score of Mettl3^fl/fl and Mettl3^CKO mice at the indicated times following SCI; ***p < 0.001. H, Footprint analysis of Mettl3^fl/fl and Mettl3^CKO mice 28 d after SCI; ***p < 0.001. I, Rotarod test of Mettl3^fl/fl and Mettl3^CKO mice 28 d after SCI; **p < 0.01. J, Ladder walking (regular- and irregular-spaced rungs) of Mettl3^fl/fl and Mettl3^CKO mice at the indicated time after SCI; ***p < 0.001. K, Representative images and quantification of MEP amplitude and latencies in Mettl3^fl/fl and Mettl3^CKO mice at day 28 postinjury; ***p < 0.001. L, Representative immunofluorescence images of GFAP and CD68 expression and quantification of lesion area and CD68⁺ macrophages/microglia in Mettl3^fl/fl and Mettl3^CKO mice at day 14 postinjury; ***p < 0.001. M, Double staining of GFAP and Ki67 and quantification of GFAP⁺Ki67⁺ cells in spinal cords of Mettl3^fl/fl and Mettl3^CKO mice at day 14 postinjury; ***p < 0.001. N, Immunofluorescence images of GFAP and CSPG4 and quantification of CSPG4⁺ area in spinal cords of Mettl3^fl/fl and Mettl3^CKO mice; ***p < 0.001. O, Immunostaining of NeuN in spinal cords of Mettl3^fl/fl and Mettl3^CKO mice at day 14 postinjury and quantification of viable neurons in Z1–Z4 zones adjacent to lesion boundary; ***p < 0.001. P, Double staining of GFAP and NF in spinal cords of Mettl3^fl/fl and Mettl3^CKO mice at day 28 postinjury and quantification of NF⁺ axons at various distances from lesion center; ***p < 0.001. Two-way ANOVA followed by post hoc Bonferroni correction (E, G, J, O, P); Student’s two-tailed unpaired t test (C, D, F, H, I, K, L, M, N). Scale bar, 200 µm.

YAP1 is a potential target of METTL3 in astrocytes. A, Venn plot of different genes in MeRIP-seq and RNA-seq comparing primary astrocytes with or without METTL3 depletion. B, M⁶A peak distribution in indicated regions of primary astrocytes with or without METTL3 depletion. C, M⁶A motif of astrocytes with or without METTL3 silencing. D, Gene ontology analysis of differentially m⁶A modified genes following METTL3 knockdown. E, KEGG analysis of differentially expressed genes after METTL3 depletion. F, G, Expression and quantification of YAP1 in Mettl3^fl/fl and Mettl3^CKO mice with or without injury; *p = 0.047, **p = 0.0083, ***p = 0.0010. H, I, Expression and quantification of astrocytic total and nucleic YAP1 at indicated times after scratch injury; *p = 0.0145, ***p < 0.001. J, K, Heat maps of a set of proliferative YAP1 target genes in GSE5296 (J) and GSE42828 (K). L, Volcano plot of genes with a significant change in both m⁶A level and gene expression level after METTL3 silencing. M, The abundance of m⁶A sites in YAP1 mRNA of primary astrocytes with or without METTL3 ablation. N, MeRIP-PCR analysis of primary astrocytes precipitated with anti-m⁶A and then evaluated by real-time PCR of YAP1; ***p < 0.001. O, RIP-PCR analysis of primary astrocytes precipitated with anti-METTL3 and then evaluated by real-time PCR of YAP1; ***p < 0.001. P, MeRIP-qPCR analysis of control and METTL3 knockdown astrocytes precipitated with anti-m⁶A, and subsequently probed with YAP1; ***p < 0.001. One-way ANOVA followed by post hoc Bonferroni correction (G, I, N, O); two-way ANOVA followed by post hoc Bonferroni correction (P). Data of MeRIP sequence comparing astrocytes with or without METTL3 depletion are shown in Extended Data Figure 7-1.

METTL3 regulates the stability of YAP1 mRNA in an IGF2BP2-dependent manner. A, Relative mRNA expression of astrocytic YAP1 and METTL3 following METTL3 knockdown; ***p < 0.001. B, Expression and quantification of total YAP1 and nucleic YAP1 after METTL3 silencing; ***p < 0.001. C, Half-life of astrocytic YAP1 mRNA with or without METTL3 depletion was evaluated by RT-qPCR; ***p < 0.001. D, Relative mRNA level of YAP1 after METTL3 overexpression with or without 1 µm methylation inhibitor treatment for 24 h; ***p < 0.001. E, Expression and quantification of total YAP1 and nucleic YAP1 after METTL3 overexpression with or without 1 µm methylation inhibitor treatment for 24 h; ***p < 0.001. F, Schematic diagram of luciferase reporter plasmid. G, Effects of METTL3 knockdown on luciferase activity of astrocytes transfected with WT or YAP1 m⁶A sites mutated luciferase reporter plasmid; ***p < 0.001. H, Half-life of astrocytic YAP1 protein with or without METTL3 depletion was evaluated and quantified by Western blotting; p = 0.2615. I, Ubiquitination levels of YAP1 protein in control and METTL3-silenced astrocytes were detected by Co-IP analysis. J, Ubiquitination levels of YAP1 protein in Mettl3^fl/fl and Mettl3^CKO mice with or without SCI were examined by Co-IP. K–M, Correlation analysis between YAP1 and m⁶A-related genes based on GSE5296 and GSE42828. N, O, Relative mRNA expressions of IGF2BP2 in GSE5296 (N) and GSE42828 (O); *p = 0.047, **p = 0.0013, ***p < 0.001. P, Expression of IGF2BP2 protein at the indicated times after SCI was detected by Western blot. Q, Relative mRNA expression of IGF2BP2 in RNA sequence of scratch-injured astrocytes; **p = 0.0035. R, Expression of astrocytic IGF2BP2 protein at the indicated times after scratch injury. S, Relative mRNA expression of astrocytic YAP1 and IGF2BP2 following IGF2BP2 knockdown; ***p < 0.001. T, Expression and quantification of total YAP1 and nucleic YAP1 after IGF2BP2 silencing; ***p < 0.001. U, Half-life of astrocytic YAP1 mRNA with or without IGF2BP2 depletion was evaluated by RT-qPCR. p = 0.0045. V, Relative mRNA level of YAP1 after IGF2BP2 overexpression with or without 1 µm methylation inhibitor treatment for 24 h; **p < 0.01, ***p = 0.0008. W, Expression and quantification of total YAP1 and nucleic YAP1 after IGF2BP2 overexpression with or without 1 µm methylation inhibitor treatment for 24 h; *p < 0.05, ***p < 0.001. X, Half-life of astrocytic YAP1 protein with or without METTL3 depletion was evaluated after 10 µg/ml CHX treatment and quantified by Western blotting. p = 0.4245. Y, Ubiquitination levels of YAP1 protein in control and IGF2BP2-silenced astrocytes were detected by Co-IP assay. One-way ANOVA followed by post hoc Bonferroni correction (A, B, D, E, G, N, O, S, T, V, W); Two-way ANOVA followed by post hoc Bonferroni correction (C, H, U, X); Nonlinear regression of one phase decay (C, H, U, X); Student’s two-tailed unpaired t test (Q); Pearson correlation analysis (K–M).

METTL3 promotes functional recovery and reactive astrogliosis via upregulating YAP1 expression after SCI. A, Specific expression of AAV-YAP1 was verified by immunofluorescence staining GFAP and Flag. B, Protein levels of GFAP, YAP1, and METTL3 in indicated groups were examined and quantified via Western blotting; *p < 0.05. C, BMS score of Mettl3^fl/fl and Mettl3^CKO mice injected with or without AAV-YAP1 at the indicated time after SCI; *p < 0.05. D, Rotarod test of mice in indicated groups at day 28 postinjury; *p < 0.05, **p = 0.0083, ***p < 0.001. E, Representative images and quantification of MEPs amplitude and latencies of mice in indicated groups at day 28 postinjury; *p < 0.05, **p = 0.0015, ***p < 0.001. F, Sensory behaviors of mice in indicated groups at day 28 postinjury were assessed by von Frey assay; *p = 0.0492, ***p < 0.001. G, At day 14 postinjury, double staining of GFAP and CD68 in spinal cords of Mettl3^fl/fl and Mettl3^CKO mice injected with or without AAV-YAP1 and quantification of lesion area and CD68⁺ macrophages/microglia; *p = 0.0164, **p < 0.01, ***p < 0.001. H, Immunostaining of GFAP and CSPG4 in spinal cords of the indicated groups. I, Double staining of GFAP and Ki67 in spinal cords of the indicated groups at day 14 postinjury. J, Immunostaining of NeuN in spinal cords of indicated groups at day 14 postinjury and quantification of viable neurons in Z1–Z4 zones adjacent to lesion boundary; **p < 0.01, ***p < 0.001. K, Double staining of GFAP and NF in spinal cords of indicated groups at day 28 postinjury and quantification of NF⁺ axons at various distances from lesion center; ***p < 0.001. One-way ANOVA followed by post hoc Bonferroni correction (B, D–G). Two-way ANOVA followed by post hoc Bonferroni correction (C, J, K). Scale bar, 200 µm.

METTL3-mediated reactive astrogliosis and functional recovery after SCI are dependent on its m⁶A methyltransferase function. A, Protein levels of GFAP, YAP1, and Flag in indicated groups were examined and quantified by Western blotting; **p < 0.01. B, BMS score of Mettl3^CKO mice injected with AAV-METTL3-WT or AAV-METTL3-MUT at the indicated time after SCI; ***p < 0.001. C, Rotarod test of mice in indicated groups at day 28 postinjury; *p < 0.05, **p = 0.0087. D, Representative images and quantification of MEP amplitude and latencies of mice in indicated groups at day 28 postinjury; *p = 0.0201, **p < 0.01, ***p = 0.0003. E, Sensory behaviors of mice in indicated groups at day 28 postinjury were assessed by von Frey assay; **p = 0.002, ***p < 0.001. F, At day 14 postinjury, double staining of GFAP and CD68 in spinal cords of Mettl3^CKO mice subjected to AAV-METTL3-WT or AAV-METTL3-MUT injection and quantification of lesion area and CD68⁺ macrophages/microglia; **p < 0.01, ***p < 0.001. G, Immunostaining of GFAP and CSPG4 in spinal cords of the indicated groups. H, Double staining of GFAP and Ki67 in spinal cords of the indicated groups at day 14 postinjury. I, Immunostaining of NeuN in spinal cords of indicated groups at day 14 postinjury and quantification of viable neurons in Z1–Z4 zones adjacent to lesion boundary; *p < 0.05, ***p < 0.001. J, Double staining of GFAP and NF in spinal cords of indicated groups at day 28 postinjury and quantification of NF⁺ axons at various distances from lesion center; ***p < 0.001. One-way ANOVA followed by post hoc Bonferroni correction (A, C–F). Two-way ANOVA followed by post hoc Bonferroni correction (B, I, J). Scale bar, 200 µm.