Fig 2: MEP50/PRMT5 complex induces GLI1 methylation. a, b Methylation of GLI1 in MEP50- (a) or PRMT5- (b) knockdown C3H10T1/2 cells. siMEP50-m2 and siPRMT5-m2 siRNAs were stably expressed by recombinant retroviruses. Cells transfected with FLAG-GLI1 were cultured for 24 h, followed by treatment with 300 nM SAG for 24 h. Methylated GLI1 was detected by immunoprecipitation with an anti-FLAG antibody followed by immunoblot with anti-SYM11 antibody. c In vitro methylation assays to determine the region including methylated arginine residues in GLI1 deletion mutants. HA-PRMT5 expression plasmid was transfected into HEK293T cells. At 48 h after transfection, the cells were lysed, and HA-PRMT5 was immunoprecipitated using an anti-HA (3F10) antibody. GST-GLI1 deletion mutants coupled to glutathione sepharose were incubated with immunoprecipitated HA-PRMT5 from HEK293T cells. Upper panel represents the methylated GST-GLI1 deletion mutant. Lower panel represents 20% input of GST-GLI1 deletion mutants detected by CBB R-250 staining. HA-PRMT5 expressed in 10% of total lysate used for immunoprecipitation is shown in the right panel. d In vitro methylation assays to determine methylation sites in GLI1 using amino acid substitutions (arginine to lysine) of candidate methylation sites. In vitro methylation assays were performed as described in (c). Upper panel represents methylated GST-GLI1 mutants. Lower panel represents 20% input of GST-GLI1 mutants detected by CBB R-250 staining. Underlined text denotes highly conserved residues among mammals, as shown in Supplementary Fig. 4. In c, data represent one of three independent experiments with similar results. In a and d, data represent one of twice independent experiments with similar results. Unprocessed original scans of blots are shown in Supplementary Fig. 6

Fig 3: GLI1 interacts with the MEP50/PRMT5 complex. a FLAG-GLI1 interacted with endogenous MEP50 and interaction of FLAG-GLI1 and MEP50 was increased by HH signalling pathway activation. C3H10T1/2 cells were transfected with FLAG-GLI1 or the empty vector for 24 h and then treated with 300 nM SAG for an additional 24 h. Interaction of FLAG-GLI1 and MEP50 was detected by immunoprecipitation with anti-FLAG antibody followed by immunoblot analysis using anti-FLAG and anti-MEP50 antibodies. b Schematic structures of MEP50 deletion mutants. c Mapping of the GLI1-binding region in MEP50 by immunoprecipitation analysis. HEK293T cells were transfected with Myc-MEP50 deletion mutants and FLAG-GLI1 plasmids for 24 h. Interaction of FLAG-GLI1 and Myc-MEP50 deletion mutants was detected by immunoprecipitation with anti-FLAG antibody followed by immunoblot analysis using anti-FLAG and anti-Myc antibodies. d Schematic of GLI1 deletion mutants. e GST pull-down assays to map the MEP50-binding region in GLI1. GST-GLI1 deletion mutants coupled to glutathione sepharose were incubated with immunoprecipitated Myc-MEP50 from HEK293T cells. Immunoblotting was performed with an anti-Myc antibody. In a and e, data represent one of three independent experiments with similar results. In c, data represent one of two independent experiments with similar results. Unprocessed original scans of blots are shown in Supplementary Fig. 6

Fig 4: PRMT5 regulates the proliferation of laryngeal carcinoma via the Wnt signaling pathway.a Heatmap representing gene expression changed between shPRMT5 and shRNA ctrl cells. Red and blue indicate high and low mRNA expression, respectively. b Volcano plot representing gene expression differences in PRMT5-slienced Tu686 cells. Each gene is represented by a dot with the red or blue dots indicating the differentially expressed transcripts that are statistically significant. c KEGG enrichment analysis showed the distribution of terms exhibiting statistically significant differences. d The relative mRNA levels of the indicated genes were normalized to the GAPDH level in the Tu686 cells stably transfected with control or shPRMT5 as determined by qRT-PCR. The results are expressed as the mean + SD of three independent experiments. e Luciferase reporter assay for its activity of overexpressing PRMT5 increased Topflash reporter activities in Tu212 cells. f Knockdown of PRMT5 impaired Topflash reporter activities in Tu686 cells. g The expression of β-catenin (nuclear and total) was determined by western blotting in PRMT5-knockdown Tu686 cells. h, i Wnt/β-catenin pathway inhibitor XAV939 or activator LiCl dramatically suppressed or restarted cell proliferation of PRMT5 mediated, respectively. j Pearson correlation analysis was performed between PRMT5 and β-catenin expression in our first cohort of samples. Data are the mean ± SD of three experiments. *p < 0.05, **p < 0.01.

Fig 5: MEP50/PRMT5 complex-mediated GLI1 stabilisation enhances Gli transcriptional activity and HH signalling pathway activation induces PRMT5 and MEP50 expression. a Gli transcriptional activity in PRMT5 or MEP50 knockdown cells. siMEP50-m2 and siPRMT5-m2 siRNAs were stably expressed by recombinant retroviruses. A multimerized Gli-binding site luciferase reporter plasmid and phRL-TK control reporter plasmid were transfected into C3H10T1/2 cells. After 24 h of incubation, 300 nM SAG was applied for 24 h, and then luciferase assays were performed. b qRT-PCR analysis of Ptch1, Bcl2, and Foxm1 expression in C3H10T1/2 cells with MEP50 knockdown or PRMT5 knockdown and treated with 300 nM SAG for the indicated times. siMEP50-m2 and siPRMT5-m2 siRNAs were stably expressed by recombinant retroviruses. c Gli transcriptional activity in HA-PRMT5 or Myc-MEP50-expressing cells. HA-PRMT5, HA-PRMT5 G367A/R368A, or Myc-MEP50 and a multimerized Gli-binding site luciferase reporter plasmid and phRL-TK control reporter plasmid were transfected into C3H10T1/2 cells. After 24 h of incubation, 300 nM SAG was applied for 24 h, and then luciferase assays were performed. d qRT-PCR analysis of Ptch1, Bcl2, and Foxm1 expression in HA-PRMT5 or Myc-MEP50-expressing C3H10T1/2 cells. HA-PRMT5, HA-PRMT5 G367A/R368A, or Myc-MEP50 plasmids were transfected into C3H10T1/2 cells. After 24 h of incubation, cells were separated equally, and DMSO (−) or 300 nM SAG (+) were applied for 24 h. Protein levels are shown in Supplementary Fig. 3a. e and f qRT-PCR analysis of PRMT5 (e) and MEP50 (f) mRNA expression in C3H10T1/2 cells after 24 h of treatment with 300 nM SAG. In a–c, data represent one of two independent experiments with similar results. In e and f, data represent one of three independent experiments with similar results. The source data is shown in Supplementary Data 1