(Figure 4, lanes 1–5). Three major sizes of shift were observed for both wild-type and mutant B4 HIPK3-T exons. Very slightly less strong shifts at low Tra2b con- centrations were observed after single ESE disruption (lanes 6–10), indicating that HIPK3-T exons lacking single ESEs are still able to bind Tra2b efﬁciently. Consistent with expectations from the AGAA-rich ESE elements, less RNA was shifted for probes containing double (B5, Figure 4A, lanes 21–25) and particularly triple ESE mutations (B6, Figure 4A, lanes 11–15). Indeed, at the lower concentrations of Tra2b protein tested (10 and 20 ng) a signiﬁcant part of the B5 and B6 RNA remained un-shifted, and the highest molecular weight band shift was not observed in the B6 mutant even with the highest concentration of Tra2b (lane 15 compared to 5). We also compared the binding of wild-type HIPK3-T to mutant RNAs with double ESE disruptions (Figure 4C, wt and B5) by pull down from nuclear extracts. No dif- ference in binding of Tra2b relative to ASF/SF2 was observed for any of these RNAs. Taken together these data indicate that each of the identiﬁed putative ESEs are able to interact with Tra2b protein, and mutation of one (B4) or two (B5) ESEs does not dramatically change the interaction properties of the exon. However, while B5 and wild-type RNAs form similar sized protein–RNA complexes, in vivo only the wild-type RNA sequence supports splicing activation by Tra2b. Splicing of HIPK3-T exon variants with stronger splice sites need fewer ESEs for splicing activation, but activate with lower amplitude Taken together the above experiments indicated that while in vivo splicing activation of the HIPK3-T exon is severely compromised after loss of only a single ESE sequence, Tra2b protein is still able to bind to HIPK3-T RNA in vitro efﬁciently after single ESE disruption. Hence although Tra2b binding might take place to a reduced complement of ESEs, four functional ESEs are the minimum required to enable splicing activation of the wild-type HIPK3-T exon by Tra2b. We further tested whether a combination of three ESEs, although non-functional in the wild-type context of exon HIPK3-T, might retain some splicing enhancer activity by analysing splicing of engineered versions of HIPK3-T with stronger splice sites. ESE2 was mutated in the context of both the intermediate (to give C1) and strong 50-splice site (to give C2) derivatives of HIPK3-T. We then tested each of these engineered HIPK3-T exon variants for splicing activation by Tra2b (Figure 5). Consistent with Tra2b still binding to ESE-disrupted HIPK3-T exons, ESE2 removal reduced rather than prevented Tra2b-mediated splicing in the context of an improved 50-splice site (Figure 5B and C, Columns 3–6). As previously observed, pre-mRNAs made from B3 were not activated for splicing by Tra2b (Figure 5B, Columns 1–2). Analysis of this data by plotting the difference in the log-score of splicing activation before and after co-transfection with Tra2b in Figure 5D again showed a clear correlation between the quantitative response to Nucleic Acids Research, 2011, Vol. 39, No. 18 8099 Figure 5. HIPK3-T exon variants with stronger splice sites have a reduced ESE content requirement for splicing activation, but are activated with a lower amplitude. (A) Organisation of pre-mRNAs used in this experiment. Splicing responses of HIPK3-T exon variants B3, C1 and C2, described fully in Table 1, are shown in terms of percentage splicing inclusion in response to Tra2b (B), Log-score plot for response to Tra2b (C), or difference in log scores (D). Tra2b and the overall intrinsic strength of the exon. The pre-mRNA with the intermediate 50-splice site (C1) showed a strongly increased responsiveness to Tra2b after ESE2 mutation (compare the log score plot for C1 in Figure 5 to wt and A1 in Figure 2), although splicing in- clusion levels were reduced comparatively to the wild-type HIPK3-T substrate. This was exactly the trend described in the previous section for the wild-type combination of four ESEs. HIPK3-T variants with the strong 50-splice site that either contained an ESE2 mutation (C2), or not (A2) showed very similar behaviour showing these pre-mRNAs are less dependent on Tra2b for splicing inclusion (compare A2 and C2 in Figures 2 and 5 middle and bottom panels). Identical results were also obtained on combining another single ESE disruption mutant (B4) with the intermediate and strong 50-splice site mutants (mutations C3 and C4, Table 1, and data not shown).