(Model 2 in Figure 6A). In this case binding of Tra2b to only one ESE may be sufﬁcient for activation. Thus increasing the nuclear concentration of Tra2b might com- pensate for the decreased ESE content by increasing the probability of ESE occupancy. In order to test between the predictions of these two models, we carried out transfections with a gradient of different Tra2b concentrations. Splicing of the wild-type HIPK3-T exon was efﬁciently activated in response to increasing Tra2b-GFP. Although B1 or B3, with three of four ESEs remaining, showed a minimal response to Tra2b, only very low levels of exon inclusion are achieved (Figure 6), and thus even high levels of Tra2b cannot compensate for an incomplete set of ESEs even in very high concentrations of Tra2b. Western analysis of transfected cells is shown in Figure 6C, which shows the increase in Tra2b-GFP along the concentration gradient, and also the levels of endogenous Tra2b protein and alpha-tubulin in the same cells. Splicing repression of a strong downstream 50-splice site enables the HIPK3-T splicing switch to operate The above data indicate that a combination of four ESEs coupled to a weak 50-splice site enable an efﬁcient on/off splicing switch in response to increasing concentrations of Tra2b. However, proper operation of the HIPK3-T splicing switch system would require either any nearby competing stronger 50-splice sites not dependent on Tra2b activation to be actively repressed, or for the Tra2b protein to activate splicing at the upstream 50-splice site. Sequence analysis indicated that immediately down- stream of HIPK3-T is a strong 50-splice site consensus sequence that is in fact not selected by the spliceosome in vivo (19). This sequence has a score of 6.25 compared to the natural 50-splice site which only measures 0.16 using a maximum entropy model (http://genes.mit.edu/ burgelab). To test whether this downstream splice site is actively repressed, or whether Tra2b preferentially acti- vates the upstream 50-splice site irrespective of intrinsic splice site strength, we ﬁrst removed the weak upstream natural 50-splice site (mutant A3). In this case we only observed very weak splicing at the downstream site (Figure 7, Columns 3 and 4). This indicates that the upstream natural 50-splice site is indeed preferentially activated by the Tra2b sites in the exon, but the down- stream splice site is functional at some level since it is activated by Tra2b when the upstream weaker site is absent. To test for active repression of the downstream site we then replaced the natural 50-splice site with the down- stream stronger 50-splice site, by simply deleting 37 nt off the end of the exon in place of the weak natural 50-splice site (mutant A4). In this case we observed no splicing in- clusion even with transfected Tra2b (Figure 7, Columns 5 and 6). This conﬁrms that this downstream 50-splice site region is also actively repressed, and moving the repressive elements closer to the HIPK3-T exon may repress splicing inclusion. Consistent with this model, the downstream Nucleic Acids Research, 2011, Vol. 39, No. 18 8101 Figure 7. The stronger 50-splice site downstream of the HIPK3-T exon is repressed. (A) Organization of the different pre-mRNAs used in this experiment. (B) Percentage of splicing inclusion of each of the engin- eered HIPK3-T exons at the downstream splice site in response to co-transfection of either GFP or Tra2b-GFP. (C) Splicing inclusion of each of the HIPK3-T exon derivativesmonitored by a log-score plot. intronic region binds a high concentration of hnRNPs, but not SR proteins (Figure 4C). DISCUSSION Alternative splice site selection is regulated by ubiquitous- ly expressed splicing factors that act on sequences located within or in the vicinity of exons. This gives rise to at least two variables for each exon, namely, the complement of splicing factors available in the target tissue, which is often controlled within very narrow concentration ranges; and secondly, the intrinsic strength of the exon reﬂected by its splice site strengths and complement of positive and negative regulatory elements. The recognition of an exon is based on a ﬁnely regulated balance between these two variables. It has been a conceptual challenge in the ﬁeld to explain how individual exons are regulated in a tissue or cell speciﬁc manner in response to ubiquitously expressed factors. We present here evidence for an interesting and novel splicing switch mechanism, that relies on a balance between the number of binding sites for a single splicing factor coupled to 50-splice site strength. In the HIPK3-T system, a very weak exon which is not recognized in any tissue type tested other than testis contains an array of AGAA-rich ESEs and is activated by Tra2b. Although the HIPK3-T exon binds in nuclear extracts to a number of splicing regulator proteins in addition to Tra2b (19),