Fig 1: CNOT7v2 promotes CD44 exon inclusion. (A) A pie chart showing the distribution of CNOT7-regulated cassette exon events. (B) Exon structure of the pre-mRNA encoding the transmembrane protein CD44, with the nine constitutive (black boxes) and nine variable exons (white boxes). Paired arrows indicate primer sets to amplify CD44 v5/v6, v8/v9 and c2/c3 containing isoforms. (C) qRT-PCR of CD44 variable mRNA normalized against the constitutive CD44c2/c3 expression level in non-transfected (mock), non-targeting control (siCTL) and CNOT7v1- or v2-knockdown MCF7 cells. Ratios of CD44 isoforms (CD44v5/v6, and CD44v8/v9) between CNOT7 knockdown cells and control cells were plotted. (D,top) Schematic diagram of the CD44v8 minigene construct. The v8 exon and its flanking introns are inserted between two constitutive exons as described in (33). Paired arrows indicate qPCR primer sets used to quantify both skipping and inclusion events. (Bottom) qRT-PCR analysis of RNA harvested from MCF7 cells transfected with the v8 minigene showing that CNOT7v2, and CNOT7v1 to a lesser extent, promotes CD44 exon inclusion. Ratios of v8 exon inclusion and skipping were plotted. (E) CNOT7 proteins affect the inclusion of the CD44 v5-exon sequence. (Top) A schematic representation showing the splice-reporter gene with insulin exon sequences (open boxes), CD44 v5 exon (black box), Luciferase (Luc) coding sequence and introns (black lines); v5 exon skipping (gray) and inclusion (black) events are indicated. The lower panels show v5-luciferase splice-reporter activity of HEK293T cells co-transfected with pCI-V5-CNOT7v1/2 or pCI vectors (left panel). Two amounts of V5-tagged CNOT7 encoding pCI vectors were co-transfected in HEK293T cells with the CD44 v5-Luc minigene and a control Renilla Luciferase-encoding plasmid. Firefly luciferase luminescence was normalized against Renilla Luciferase activity. (Right panel) CD44 v5-Luc expression was measured in mock, siCTL and siCNOT7v1 or v2 transfected MCF7 cells. (F) Immunoblot analysis showing an increase in the expression of CD44 variants (CD44v) and a decrease in CD44 standard (CD44s) splice isoform level upon knockdown of CNOT7 isoforms in MCF7 cells. LE, long exposure; SE, short exposure. The extracts were first analysed by western blotting with an anti-CD44 antibody. The membrane was then stripped and reprobed with an anti-CD44v5. Representative of three independent experiments. (G) Bar graph, obtained by densitometric analysis of western blot data (F), shows quantified intensities of the CD44s and CD44v5 bands normalized against a-tubulin. The experiments illustrated in (C), (D), (E) and (G) were performed in triplicate, expressed as mean values (±SD) of three independent experiments. The P-value was determined by two-tailed Student's t-test using the ‘siCTL’ condition as the reference population: (ns) non-significant, (*) P < 0.05; (**) P < 0.01; (***) P < 0.001. The efficiency of the siRNA knockdown was verified by qPCR (see Supplementary Figure S2F and S2G).