Fig 2: PTBP1 bound exon 30 and adjacent intronic areas of ITSN1 RNA and modulated splicing of ITSN1.A In vitro RNA pull-down assay in LN229 cell line. Proteins were subjected to western blot analysis with anti-PTBP1 antibody after pull-down. B Analysis of PTBP1 CLIP-seq data from GSE19323 and GSE114720, the blue lane shows PTBP1 CLIP peaks for exon 30 and adjacent area of introns of ITSN1. C The primer position used to detect the binding segment of PTBP1 and ITSN1 pre-mRNA in the RIP experiment. D PTBP1 protein precipitation upon RIP with anti-PTBP1. E ITSN1 amplification from the anti-PTBP1 coprecipitated RNA fraction. F RIP with anti-PTBP1 followed by RT–qPCR quantification, values were expressed as mean ± SD from three independent experiments (two-tailed Student’s t test, **P < 0.01, ***P < 0.001). G The potential binding sequences of PTBP1 within ITSN1 E30 RNA used in the RNA pulldown assay and its position in the ITSN1 pre-mRNA, the red font represented the mutated base. H RNA pulldown assay was used to detect the binding of PTBP1 to the biotinylated ITSN1 E30 WT RNA and ITSN1 mutant RNA. I Schematic representation of the ITSN1 minigene and the primer sets for PCR detection of ITSN1 exon 30-included and -excluded products. The primers for the internal reference of exogenous ITSN1-minigene were CMV-F and E29-R. The mutated base sequence of the ITSN1-minigene was marked with red font. J Western blot analysis for the inhibition of PTBP1 in transient cells transfected with the wild-type and mutant ITSN1-minigene plasmids. K Representative pictures of agarose PCR gels depicting the inversion in the splicing pattern of ITSN1 regulated by PTBP1 after transfected with the wild-type and mutant ITSN1-minigene plasmids. L RT–qPCR analysis showing changes in the ratio of ITSN1-S/ITSN1-L upon silencing PTBP1 in LN229 cells expressing ITSN1 minigene or ITSN1 mutant minigene. Values were expressed as mean ± SD from three independent experiments (two-tailed Student’s t test, ***P < 0.001).

Fig 3: PTBP1 promoted proliferation, migration, and invasion of glioma cells by regulating alternative splicing of ITSN1 in vitro.A Establishment and verification of stable cell lines silencing PTBP1 followed by knocking down of ITNS1-L, Values were expressed as mean ± SD from six independent experiments (two-tailed Student’s t test, ***P < 0.001). B Images of EdU staining (left, scale bars, 100 µm) and the comparison of EdU-positive rates among indicated cells (right). C Migration assay of the indicated cells. Cells migrating through transwell inserts were stained, photographed, and quantified. Scale bars, 200 µm. D Invasion assay results. Cells invading through matrigel matrix-coated transwell inserts were stained, photographed, and quantified. Scale bars, 200 µm. E Western blot analysis of stable cell lines silencing PTBP1 followed by overexpression of ITSN1-S. F Images of EdU staining (left, scale bars, 100 µm) and the comparison of EdU-positive rates among indicated cells (right). G Migration assay of the indicated cells. Cells migrating through transwell inserts were stained, photographed, and quantified. Scale bars, 200 µm. H Invasion assay of the indicated cells. Cells invading through matrigel matrix-coated transwell inserts were stained, photographed, and quantified. Scale bars, 200 µm. Values were expressed as mean ± SD from three independent experiments in (B–D) and (F–H) (two-tailed Student’s t test, *P < 0.05, **P < 0.01, ns, no significance).

Fig 4: RNA distribution in polysome fractions obtained from cells overexpressing SINEUP RBPs. (A) Representative polysome gradient profile with optical density (290 nm). Fractions and correspond to the 15% and 45% sucrose fractions, respectively. (B) Polysome profiling of EGFP mRNA (green) and SINEUP-GFP RNA (blue). RNA distribution was quantified by RT-qPCR. Each fraction of EGFP mRNA in cells co-transfected with EGFP and SINEUP-GFP vectors (d–f) was compared with the corresponding fraction in cells transfected with EGFP vector alone (a–c). *P < 0.05, **P < 0.01 by Student's t-test. Data are means ± SD from three independent experiments. Fractions of SINEUP RNA in cells co-transfected with EGFP and SINEUP-GFP vectors are shown in g-i, and those in cells transfected with SINEUP RNA alone are shown in m. The protein distribution in polysome fractions with non-/overexpressed PTBP1 or HNRNPK were tested (j-m). Equal volumes of solution were pooled as Free/40S (Free or 40S binding RNA fraction from – in A), Mono (monosome fraction from –), Light (light polysome fraction from –), and Heavy (Heavy polysome fraction from –) and were applied to 10% SDS gels to detect each target protein. Western blotting images representative of least three independent experiments are shown.

Fig 5: PTBP1 promoted glioma progression.A PTBP1 expression was evaluated between LGG or GBM and normal samples by GEPIA. B Scatter plot comparing PTBP1 mRNA levels between different grades of glioma using the data from CGGA. C Curves of Kaplan–Meier analysis. OS was analyzed according to the PTBP1 expression (cut-off at 50% of the entire group). Low PTBP1 expression was related to long OS (P < 0.001). D Western blot analysis of PTBP1 knockdown and rescue in the extracts of cells as indicated. Loading control: ß-actin. E Images of EdU staining (left, scale bars, 100 µm) and the comparison of EdU-positive rates between the control and silencing PTBP1 cells (right). F Migration assay of the indicated cells. Cells migrating through transwell inserts were stained, photographed, and quantified. Scale bars, 200 µm. G Invasion assay results. Cells invading through matrigel matrix-coated transwell inserts were stained, photographed, and quantified. Scale bars, 200 µm. Values were expressed as mean ± SD from three independent experiments in (E–G) (two-tailed Student’s t test, *P < 0.05, **P < 0.01, ***P < 0.001). H Nude mice injected with control or silencing PTBP1 LN229 cells. The images shown represent results for 7 mice. Red and blue dotted lines indicated the location and size of the tumors. I Growth curve of nude mice tumors within 7 weeks, values were expressed as mean ± SD (two-way ANOVA, ***P < 0.001). J The representative images show the xenograft tumors isolated from nude mice at the 7th week of tumor inoculation. K Representative images of hematoxylin-eosin staining (left), scale bars, 200 µm. The frequency of xenograft invasion in mice was analyzed quantitatively (right), P value was calculated using the chi-square test. ***P < 0.001. L Immunohistochemical images of tumor sections stained for Ki67 from control or PTBP1 knock down mice (left), quantitation of the percentage of Ki67-positive cells in tumor sections (right). Values are mean ± SD, two-tailed Student’s t test, ***P < 0.001. Scale bars, 200 µm.