8094 Nucleic Acids Research, 2011, Vol. 39, No. 18 Figure 1. Modular organization of the HIPK3-T exon and activation by Tra2b through direct association with RNA. (A) z-score plot predicting the splicing control sequences in and around the HIPK3-T exon. Regions of the exon above the threshold for exonic splicing enhancers are labelled ESE1-4, and the region of the exon below the threshold for splicing silencers is labelled ESS. The architecture of the HIPK3-T exon and surrounding intron, and sequence of the HIPK3-T exon and ﬂanking intron sequences are shown underneath. The strong 50-splice site downstream of HIPK3-T is underlined, and its position in the sequence marked by an asterisk. (B) Schematic representation of Tra2b protein domains. (C) Analysis of the splicing response of wild-type HIPK3-T to full length and domain deleted Tra2b-GFP fusion proteins, assayed by RT–PCR and capillary gel electrophoresis, and presented as the percentage inclusion of HIPK3-T. All transfections and subsequent RNA analyses were performed at least in triplicate. (D) Example of RT–PCR from cells transfected with HIPK3-T and different expression constructs encoding Tra2b proteins, and analysed by capillary gel electrophoresis. +T = size of RT–PCR product including HIPK3-T exon. –T = size of RT–PCR product excluding HIPK3-T exon. (E) Expression levels of each of the different wild-type and domain deleted Tra2b-GFP fusion proteins, assayed by western blotting from transfected HEK293 cells in parallel with splicing assays. Levels of GFP fusion protein were measured using an a-GFP antibody, and levels of actin were measured on the same ﬁlter using an a-actin antibody to provide a loading control for each lane. described (25), except that the Flag-Tra2b protein was immunopuriﬁed on an anti-Flag-M2 afﬁnity gel and eluted by competition with FLAG peptide (SIGMA-ALDRICH). EMSAs were done as previously described (25). Antibodies Proteins were detected by western blotting using antibodies speciﬁc for GFP (Clontech 632 381 Living Colours A v monoclonal antibody), tubulin (Sigma T5168 anti-a-tubulin monoclonal antibody), and Tra2b (Abcam polyclonal antibody ab31353), and others used previously (19,26). RESULTS Splicing activation of HIPK3-T is dependent on both the RRM and RS domains of Tra2b The HIPK3-T exon has a weak 50-splice site, although there is a potentially stronger downstream 50-splice site that is not selected by the spliceosome (position indicated as an asterisk in Figure 1A). We used a pull down assay to monitor association of nuclear proteins with the HIPK3-T