SULT4A1 transcript and protein expression during cell differentiation and mouse embryonic development. (A) SH-SY5Y cells were cultured for up to 12 days in Dulbecco’s modified Eagle’s medium/F12 medium in the presence of 10 µM RA. At each time point, cells were harvested for mRNA and protein determination. RT-PCR was performed using primers located in exon 6 and exon 7. The variant transcript, which contained pseudo-exon 6p, produced a PCR product of 397 bp, whereas the WT transcript produced a PCR product of 270 bp. RT-PCR results are shown in the upper two images, whereas SULT4A1 protein is shown in the lower two images. (B) SULT4A1 mRNA and protein in hiPSCs during RA-induced differentiation. (C) Expression of SULT4A1 mRNA and protein in mouse embryonic tissue from day E8.5 to E18.5. β-actin was used as a housekeeping gene for RT-PCR and α-tubulin as a loading control for Western blots. Molecular weight markers are shown on the right of each Western blot.

Minigene splicing assay of SULT4A1 pseudo-exon 6p in SH-SY5Y cells differentiated with RA. (A) Deletion minigene assays: 10 minigene plasmids with sequential deletion of intronic segments of the approximately 1.6 kb full-length minigene were constructed and transfected into SH-SY5Y cells with and without RA treatment. Switching of the endogenous SULT4A1 transcript was used as a positive control (far right panels). The nomenclature of the deletion minigenes is based on the number of base pairs deleted from either the 5′ or 3′ ends of the full-length minigene. RA untreated (−) and treated (+) lanes are labeled and the ratio of the variant to the WT PCR product (V/WT) is shown next to the RT-PCR gels. Results are representative of duplicate experiments. (B) Sequence of the SULT4A1 gene upstream of pseudo-exon 6p (gray arrow) showing the 32-base sequence between 5′Δ406 and 5′Δ438 (red). Putative binding motifs for RNA processing factors are shown in the boxes with blue (MBNL) and green (CELF). Numbers refer to the number of bases upstream of the 6p pseudo-exon. (C) Effect of ectopic expression of the MBNL and CELF genes on splicing of the 5′Δ338 minigene construct in undifferentiated SH-SY5Y cells. Cells were transfected with each RNA splicing factor and the minigene. The upper panel shows the PCR products of the minigene transcript with the variant and WT indicated. Expression of each protein is shown as a FLAG Western blot below. (D) Effect of MBNL and CELF proteins on the splicing of endogenous SULT4A1 in undifferentiated SH-SY-5Y cells. The upper panel shows the variant and WT PCR products. Expression of each protein is shown as a FLAG Western blot below. Results are representative of duplicate experiments. Molecular weight markers are shown on the right of each Western blot. EV, empty vector.

Expression of SULT4A1, MBNL, and CELF transcripts in SH-SY5Y cells during RA-induced differentiation. (A) SH-SY5Y cells were treated with 10 µM RA for 10 days. RNA was isolated at the indicated times and amplified by RT-PCR. For MBNL-2, splice variants were detected. (B) Western blots of protein expression after 10 days of RA treatment. Quantification of protein levels is shown alongside each Western blot. Data are the mean ± S.D. (n = 3). *P < 0.05 (significant differences, t test). CTRL, control.