MBNL2 binds to the 3′UTR of VEGFA and controls secreted VEGF-A protein levels. (A) RT-PCR quantification showing different Atp2a1 exon 22 (E22) inclusion levels depending on inserted binding sites (BS) from the VEGFA 3′UTR and MBNL2 isoform 40 (MBNL2-40) or MBNL1 isoform 42 (MBNL1-42) expression in Mbnl1 and 2 double knockout MEF cells. Shown are ΔPSI values with respect to the negative control in which the binding site is deleted (see also Supplemental Fig. 15). Statistical significance is given in reference to the inclusion level observed with the natural Atp2a1 binding site. n = 3. (B) Relative amounts of MBNL1 and MBNL2 bound to the VEGFA 3′UTR RNA and calculated KD values. RNA carrying (CUG)20 repeats was used as positive control. n = 3. (C) Half-life of VEGFA mRNA. MCF-7 cells were treated with 5 µg/mL actinomycin D (ActD). Thereafter, total RNA was isolated at 2 h intervals and VEGFA mRNA levels were quantified by RT-qPCR. VEGFA mRNA level was normalized to the housekeeping gene RPLP0. n = 3. (D) Quantification of secreted VEGF-A protein levels by ELISA. Secreted VEGF-A levels were determined in the supernatants of normoxic and hypoxic A549 and MCF-7 cells and after knockdown of MBNL2. n = 4. (**) P < 0.01. (*) P < 0.05.
