FIGURE 7.

Mutation of putative SC35 binding site inhibits RA-mediated utilization of 5′ splice site II utilization on the minigene. a, schematic of the position and sequence of the putative SC35 cis-element on the pSPL3_PKCδ splicing minigene. Arrows indicate the position of primers used in PCR analysis. Putative SC35 binding site ggccaaag was mutated to tagcccaga on the minigene. b, resulting mutated minigene pSPL3_PKCδ** was transfected into NT2 cells. In separate wells, the mutated minigene pSPL3_PKCδ** was co-transfected with either 2 μg of SC35 or SF2/ASF. The original pSPL3_PKCδ splicing minigene was also transfected in a separate well. After overnight transfection, NT2 cells were treated with or without 10 μm RA for 24 h. Total RNA was extracted and RT-PCR performed using primers for PKCδ exon 10 sense and SA antisense as shown. 5% of the products were separated by PAGE and silver stained for visualization. SSI: usage of 5′ splice site I; SSII: usage of 5′ splice site II. Graph represents percent exon inclusion calculated as SS II/(SS II + SSI) × 100 and is representative of three experiments performed separately. Results indicate that mutation of the enhancer element ggccaaag abolishes the ability of RA or SC35 to promote utilization of 5′ splice site II on PKCδ splicing minigene.