Nucleic Acids Research, 2011, Vol. 39, No. 18 8097 Figure 3. Each of four Exonic Splicing Enhancer Regions (ESEs) are essential for Tra2b-mediated splicing activation. (A) The z-score plots of the wild-type HIPK3-T exon (black), superimposed with z-score plots for each of the point mutants which affected individual ESEs (shown as coloured lines, with the changed nucleotide indicated as a broken line). See also Table 1 for a complete description of all mutants used in this study. (B) The percentage exon inclusion obtained after transfection of each of these point mutants after transfection with GFP or Tra2b (+lane). Analysis of the architecture of the HIPK3-T exon To identify the splicing control elements that might mediate Tra2b-mediated HIPK3-T splicing activation we next analysed the entire exon sequence and ﬂanking intron sequences for octamers predictive of splicing enhancers and silencers based on the bioinformatic approaches of Zhang and Chasin (32–34). Detailed analysis of the result- ing octamer z-score plots indicated four distinct peaks in the HIPK3-T exon above the threshold value correspond- ing with putative Exonic Splicing Enhancers (ESEs), and a single Exonic Splicing Silencing Region (ESS) (Figure 1A). We name individual predicted ESEs here ESE 1–4 (Figure 1A). Each of the ESEs was GAA/ AGA-rich (designated hereafter AGAA-rich), consistent with the known binding site for Tra2b (15,16,35) Splicing activation of HIPK3-T by Tra2b requires the full complement of four ESEs We next sought to precisely map sequences in the HIPK3-T exon that were responsive to Tra2b. Using the results of the HIPK3-T exon sequence analysis shown in Figure 1A, we systematically mutagenized each putative Tra2b responsive ESE with multiple single mutations (Figure 3 and Table 1). In each case site directed muta- genesis was designed using an in-house software based on the octamer z-scores, along with other matrices including HSF and ESE ﬁnder (36–38) to ensure that no predicted splicing silencer or enhancer elements were introduced into the HIPK3-T sequence (see ‘Materials and Methods’ section). To our surprise, single mutation of any one of the four ESE regions was sufﬁcient to completely prevent HIPK3-T splicing activation (Figure 3B). No HIPK3-T splicing inclusion was observed for pre-mRNAs made from constructs B1, B2 (disruption of ESE1), B3, B4 (dis- ruption of ESE2), or B5 (disruption of ESE 3 and 4) (Figure 3B, Columns 1–8, Table 1 and data not shown). Further dissection of B5 into the individual point muta- tions B5x (disruption of ESE3), B5y (disruption of the ﬁrst sub-peak of ESE4) and B5z (disruption of the second sub-peak of ESE4) indicated that only single point muta- tions in ESE3 (B5x) or in the ﬁrst enhancer sub-peak of ESE4 (B5y) completely disabled Tra2b-mediated splicing activation (compare Columns 1 and 2 with Columns 9–12, Figure 3B). Disruption of the second sub-peak alone in ESE4 (B5z) only resulted in a partial loss of Tra2b-mediated splicing activation, most probably because the mutation preserves a AGAAG sequence in the ﬁrst half of ESE4 (Columns 13, 14, Figure 3B). HIPK3-T exons lacking single ESEs are still able to bind Tra2b protein The above results indicate that ESEs 1–4 as deﬁned by our approach are indeed true individual ESEs, and also each