Fig. 6: USP39 selectively regulates exon inclusion/exclusion via interaction with SRSF6 in a position-dependent manner.

A Comparison of motif enrichment analysis results between human HCC cells and mice. The depleted SRSF6-binding motif and the enriched HNRNPC-binding motif (shown on the right panel) were distributed in a conserved pattern in humans and mice. B, C RNA pulldown assay verified the binding of SRSF6 to the putative binding motif (B) and predominant binding of SRSF6 within the flanking constitutive exons over the cassette exons (C). Representative images of three independent reproducible experiments are shown. D RIP assay was performed in PLC-8024 cells with SRSF6 antibody or IgG control (n = 3). E Minigene reporters of KANK2, ZNF655, and NEK6 were introduced into USP39-knockdown SNU-449 cells. SRSF6 was further silenced and splicing of the minigenes was verified by RT-PCR. The percentages of cassette exon inclusion within the total transcripts were presented using PSI. F Schematic illustration of the mechanism by which shUSP39 represses exon inclusion through interacting with SRSF6 in a position-dependent manner. G Endogenous (left)/ ectopic overexpressed (right) USP39 was coimmunoprecipitated with endogenous (left)/ ectopic overexpressed (right) SRSF6 protein. H RIP assay showed that USP39 retained in the flanking constitutive exons upon USP39 knockdown (blue columns). Silencing of SRSF6 in USP39-knockdown cells abolished USP39 retainment in the flanking constitutive exons (red columns). Data were presented as fold enrichment between downstream exon signal and cassette exon signal (downstream/cassette). Mean ± SD. P values were determined using paired (D) or unpaired (H) Student’s t-test. *P < 0.05, **p < 0.01.