8096 Nucleic Acids Research, 2011, Vol. 39, No. 18 exon and with the downstream intronic region containing this strong 50-splice site. Consistent with the observed physiological selection of the HIPK3-T exon, the exonic region strongly bound some SR proteins and the SR-like protein Tra2b (Figure 4C). In contrast, the downstream region did not associate with SR proteins, but did bind to hnRNPs, which are frequent splicing repressors (Figure 4C). Tra2b is the only known splicing activator of HIPK3-T (19) and in preliminary experiments we found no other interacting RNA binding proteins signiﬁcantly affected its splicing inclusion. To test if direct RNA binding was required for Tra2b-mediated splicing activation of HIPK3-T, we ﬁrst inactivated the RRM with an independ- ent deletion of RNP1 and deletion of the entire RRM (Figure 1B). We then tested the activity of the resulting mutants on splicing of the wild-type HIPK3-T exon by co-transfection experiments in HEK293 cells. In contrast to the wild-type Tra2b-GFP protein, mutants affecting the RRM (DRNP1 and DRRM) were unable to stimulate HIPK3-T exon inclusion (Figure 1C, D lanes 4 and 5 compared to lane 2) indicating direct binding of Tra2b-GFP to HIPK3-T target RNA is required for splicing activation. In several other SR protein splicing regulators, multiple domains can play partially redundant functions (27–30). We also deleted the N-terminal RS-rich region to test if the N- and C-terminal RS domains of Tra2b are functionally redundant, and found that Tra2b proteins lacking the N-terminal RS domain were unable to support splicing activation of HIPK3-T (Figure 1C and D lane 3). We observed equal levels of GFP-fusion proteins by western blotting (Figure 1E) for each of the full length and deletion derivatives of Tra2b, and these localized in the nucleus [data not shown, and (31)]. The amplitude of Tra2b-mediated splicing activation of HIPK3-T is dependent on intrinsic exon strength HIPK3-T has a somewhat weak 50-splice site (GAG| gttaaa), which has a score of 0.16 measured using a maximum entropy model (http://genes.mit.edu/burgelab, where a score of 11 represents a perfect splice site). We tested whether this weak 50-splice site normally contributes to exon skipping by mutating HIPK3-T to contain an intermediate strength (GAG|gttagg, score: 4.36 for con- struct A1) or strong 50-splice site (GAG|gttagt, score: 7.15 for construct A2). Full details of all HIPK3-T mutants used in this study are given in Table 1. Both improved splice sites resulted in the HIPK3-T exon being more efﬁciently spliced even without the addition of exogenous Tra2b (compare Columns 1 with 3 and 5, Figure 2B). Therefore, the level of HIPK3-T exon inclu- sion shows a direct correlation to splice site strength, indicating that the weakness of the 50-splice site is a major negative element restricting HIPK3-T exon splicing. We next analysed the magnitude of the splicing response of the above 50-splice site variants of HIPK3-T to Tra2b expression (Figure 2B, Columns 2, 4 and 6). To compare differences between splicing responses of different pre-mRNAs over the full dynamic range we monitored Figure 2. (A) The splicing response of HIPK3-T to Tra2b correlates with intrinsic exon strength deﬁned by 50-splice site. Correlation between 50-splice site strength and splicing response of HIPK3-T 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). A1 and A2 variants have intermediate strength or strong natural 50-splice site. HIPK3-T splicing activation both as a percentage of HIPK3-T inclusion (showing overall levels of splicing in- clusion, Figure 2B), and using a natural logarithm-score plot (Log-score, Figure 2C, see ‘Materials and Methods’ section). As expected, splicing of each of the HIPK3-T exons was activated by Tra2b, but with improved overall percentage splicing inclusion for the exons with strong (A2) or intermediate (A1) 50-splice sites (Figure 3C). Importantly, plotting the difference in log-scores before and after co-transfection of Tra2b indicated a clear trend in splicing activation between pre-mRNAs: the splicing response of each of the pre-mRNAs to Tra2b was inversely correlated to 50-splice site strength (Figure 2D). Hence weaker HIPK3-T exons respond more speciﬁcally to Tra2b-mediated splicing activation.