Figure 4.

Expression of GPX1 and CCN1 is regulated by NONO-mediated pre-mRNA splicing. A Global splicing efficiency analysis at splicing sites after transfection of U251 and P3 cells with siNC and siNONO. Splicing efficiency was determined as “transread count/5' and 3' intron end first base coverage”. B The top 100 downregulated genes in the P3 sequencing data based on siNC versus siNONO. The expression data was z-transformed. C Venn plot displaying the significantly downregulated mRNAs in both U251 and P3 sequencing data. The genes with decreased splicing efficiency were selected based on splicing analysis. D Sashimi plot visualization of RNA-seq reads mapping to GPX1 and CCN1 in U251 cells in response to NONO knockdown. E Western blot analysis for GPX1 and CCN1 of U251 and P3 transfected with siNC and siNONO (n = 3). F Western blot analysis for GPX1 and CCN1 of LN229- and P3-NONO-OE or -NC (n = 3). G The schematic representation of primers designed for pre-mRNA and mRNA of GPX1 and CCN1. H qRT-PCR analysis of pre-mRNA and mRNA for GPX1 in U251 and P3 transfected with siNC, siNONO-1 and siNONO-2 (n = 3). GAPDH was used for normalization. I RNA FISH probes for pre-mRNA or mRNA were used for detection in U251 (n = 3). Scale bar = 20 μm. J The NONO binding motif in the intron of GPX1 pre-mRNA. K RNA pulldown assay with GPX1 pre-mRNA, mRNA and anti-sense pre-mRNA to detect binding with NONO in U251 (n = 3). L The RIP-PCR assay with NONO to detect GPX1 pre-mRNA and mRNA (n = 3). Input was used for normalization and IgG was used for negative control. M Representative images of RNA FISH for GPX1 pre-mRNA (green) and GPX1 mRNA (red), and immunofluorescence for NONO (blue) in U251 (n = 3). The right diagram shows the relative gray value of staining on the X-axis (AB). Scale bar = 25 μm. Data are shown as mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001.