A number of target exons respond to increasing nuclear concentrations of Tra2 (17–19). Amongst its known molecular targets, Drosophila Tra2 protein regulates a splicing switch in sex determination (20,21) and human Tra2b protein regulates a splicing switch in the tissue- speciﬁc exon HIPK3-T (19). The HIPK3-T exon is within the Homeodomain Interacting Protein Kinase 3 gene, which is implicated in apoptotic control. HIPK3-T is selected only in a single tissue, the testis, which has elevated Tra2b concentrations. The mechanisms through which ubiquitously expressed factors like Tra2b can generate splicing switches in par- ticular tissues are not well understood. To address this question, here we functionally dissected the sequences needed for splicing activation of HIPK3-T. We ﬁnd that an array of exonic splicing enhancers responsive to Tra2b, coupled to a weak 50-splice site act together as a gauge to enable splicing activation in response to increased nuclear concentrations of Tra2b. We show that this switch is deli- cately balanced by the interplay between Tra2b binding and 50-splice site strength, allowing activation of alterna- tive splicing within a narrow window, outside which the exon is either invisible to the spliceosome or activated constitutively. MATERIALS AND METHODS Computational analysis of putative ESE/ESS content of HIPK3-T and ﬂanking introns We used CisPlotter (unpublished), to compare and design HIPK3-T derivative sequences. Brieﬂy, this program computes and plots the ESE or ESS score as shown in Figure 1A. We used the list of putative ESE and ESS octamers, chose the threshold z-score levels and dealt with overlapping motifs as described previously (22). This script accepts alternative nucleotides at speciﬁed pos- itions and plots the resulting change in ESE or ESS scores in a different colour to facilitate mutagenesis design (Figure 3A). Constructs Minigene splicing constructs were based on the wild-type HIPK3-T exon, and 269 and 267-nt upstream and down- stream ﬂanking intron sequence respectively, cloned into pXJ-41 as described previously (19). Site Directed Mutagenesis of HIPK3-T was carried out as previously described (22), mutants were veriﬁed by sequencing, and then subcloned back into the parent plasmid. Deletion versions of human Tra2b protein were cloned in frame into pGFP3 using standard cloning tech- niques (19,23). Tra2bDRS1 is missing amino acids 13–97, Tra2bDRRM is missing amino acids 119–168, Tra2bDRNP1 is missing amino acids 150–168 (Figure 2). For expression of full length Tra2b, its coding sequence was inserted in a pTT3 plasmid (24), in which a Flag tag was previously inserted in the Hind III site. Nucleic Acids Research, 2011, Vol. 39, No. 18 8093 Minigene splicing analysis of HIPK3-T exon variants Tissue culture, transfections of HEK293 cells, RNA puri- ﬁcation and RT–PCR was carried out using primers and conditions as previously described (19) and the RT–PCR products were analysed by capillary gel electrophoresis. The HIPK3-T reporter construct (100 ng) was transfected with or without 300 ng of Tra2b expressing plasmid using GeneJammer (Agilent) under standard conditions for 24 h. To enable analysis of splicing patterns as well as monitoring the expression of transfected splicing factors, transfected cell cultures were split into two portions and analysed by western blotting as previously described (23). Splicing was analysed as both a percentage splicing in- clusion and a logarithmic splicing inclusion. We deﬁne log-score as the natural logarithm of the ratio of HIPK3 exon T inclusion to exclusion (logit transformation). Therefore there is a one-to-one mapping between percent- age inclusion values and log-score values. A log-score of zero denotes 50% inclusion of the HIPK3 exon T. A positive log-score denotes >50% inclusion and a negative log-score denotes <50% exon inclusion. Note that the log- score values are unbounded and can theoretically range between +/– inﬁnity. In reality they will be limited by the resolution capability of band detection, which here we have taken to be 0.1% which corresponds to a limit on the log-score value of 6.9. To compare intrinsic splicing capability of two con- structs, the difference of the two log-scores is taken. This procedure has several beneﬁts over comparing fold change of the percentage inclusion values. Since the per- centage inclusion values are bounded at 0 and 100%, fold change comparisons close to these boundaries are mislead- ing. In contrast the log-score method has been designed to treat these two ends symmetrically. For example, percent- age splicing inclusion changes from 2 to 5%; 95 to 98% or 40 to 64% each have a log-score difference of 1, whereas the percentage inclusion fold change values would be 2.5, 1.03 and 1.6 respectively. RNA afﬁnity assay and EMSA HIPK3-T exon variants or the downstream intron were cloned from the minigene splicing analysis constructs in pXJ41 vector into pBluescript, and then in vitro transcribed RNAs prepared as described (23). RNA afﬁnity assays used in vitro transcribed RNAs attached to sepharose beads to pull down proteins from nuclear extracts as previously described (19). We tested for binding of candidate RNA binding proteins based on previous results (19) and on the availability of antisera. The sequences of RNAs used for electromobility shift assays (EMSAs) included the HIPK3-T exon and 11 nt of the upstream intron for wild-type or HIPK3-T exon derivatives B4, B5 and B6. Sequences are detailed in Table 1. Full length Tra2b was expressed at high level by transient transfection of suspension 293-EBNA cells (24) using FreeStyle 293-F cells, and FreeStyle expression medium according to the recommendations of Invitrogen. After 60 h transfection, 293-F cells were pelleted and washed in PBS. Extraction and puriﬁcation of Tra2b was carried out according the protocol previously