Fig. 4. SF3B3 regulates EZH2 alternative splicing and its expression correlates with levels of the full-length EZH2.

(A) SF3B3 regulated EZH2 exon 14 inclusion is cell-type independently. EZH2 exon 14 splicing was measured by RT PCR in various cell lines (786-O, Caki-1, 293T) stably expressing sh-LacZ, sh-SFPQ, or sh-SF3B3. RT PCR was performed as described in Fig. 1B. (B) Schematic representation of splicing dual-reporter assay that mimics endogenous splicing by insertion of EZH2 genome fragment (from exon 13 to 15) harbors a point mutation in exon 14 to create stop codons. (C) The bar graph depicts the skipping of EZH2 exon 14 as measured by the firefly (F-Luc) to Renilla (R-Luc) luciferase ratio. 293T and ACHN cells were infected with lentivirus expressing sh-LacZ, sh-SFPQ, sh-SF3B3-1#, or sh-SF3B3-2#. After 3 d, these stable knockdown cell lines were transiently transfected with the splicing reporter. After transfection for 24 h, luciferase activities were measured. Data are mean ± s.d., n = 3 independent experiments, **P < 0.01. (D) SF3B3 is upregulated in renal clear cell carcinomas. Total RNA isolated from paired ccRCC tumors and adjacent normal tissues were assayed by real-time RT-PCR. * P < 0.05. (E) Positive correlation between EZH2-(14+)/EZH2 total mRNA ratio and expression levels of SF3B3 was observed in RCC samples. Relative mRNA levels of SF3B3 and the corresponding levels of EZH2 (ratio of EZH2-(14+)/EZH2 total mRNA) was plotted in each patient sample (P < 0.05, R2 = 0.4319).