8102 Nucleic Acids Research, 2011, Vol. 39, No. 18 none of these other RNA binding proteins, particularly ASF/SF2 which may also bind to a GAA-rich target sequence (23,39), could activate or repress this exon in in vivo minigene experiments (data not shown). In contrast to transfected cells, in vitro splicing substrates containing the HIPK3-T splicing enhancer do require addition of ASF/SF2 to enable splicing activation of a 50-splice site by Tra2b (19). This in vitro requirement is consistent with the need for exogenous SR proteins to support splicing of other pre-mRNA substrates in S100 extracts, since Tra2b cannot provide this additional activity. Interestingly, loss of even a single ESE sequence resulted in complete loss of exon activation which could not be rescued by elevating levels of Tra2b. An array of ESEs responsive to a single splicing factor where disrup- tion of just a single ESE results in loss of exon recognition is novel and consistent with a model in which exon strength is ﬁnely balanced with ESE content to enable an efﬁcient splicing switch to take place in the testis in response to speciﬁcally to Tra2b concentration. Such gauges may be used by exons to measure the nuclear con- centration of speciﬁc splicing regulators that are ubiqui- tously expressed but at varying tissue-speciﬁc levels, and thus establish cell type-speciﬁc splicing responses. Importantly, to enable these gauges to work properly potentially stronger consensus downstream 50-splice sites would need to be actively repressed, or else these stronger sites would be constitutively selected by the spliceosome. We have experimentally conﬁrmed repression of a poten- tially competing 50-splice site for HIPK3-T. Similar repres- sion of downstream 50-splice sites has also been observed for other regulated exons with weak 50-splice sites (40). While HIPK3-T contains multiple similar ESEs all re- sponding to Tra2b concentrations, splicing of other exons have been reported to be individually dependent on multiple different functional ESEs responsive to differ- ent RNA binding proteins (34). This suggests that a model of splicing control in which multiple ESEs respond to the nuclear concentration of speciﬁc RNA binding proteins might extend also to combinatorial control. The number of Tra2b binding sites required within an exon to establish a splicing response is likely to depend on splice site strength and other splicing signals. For example, the in- clusion of another Tra2b-responsive exon such as SMN2 exon 7, which has a single Tra2b binding site, additionally depends on binding of hnRNPG and SRp30c (15). Our analysis here of the human testis speciﬁc HIPK3-T exon makes interesting comparison to the mammalian SFRS10 exon 2 which is also regulated by Tra2b. The SFRS10 gene encodes Tra2b protein, and exon 2 is a ubiquitously spliced poison exon which enables auto- regulation in response to increasing levels of Tra2b protein (41). Similarly to HIPK3-T, SFRS10 exon 2 has 4 AGAA rich ESEs distributed all along the exon. However, SFRS10 exon 2 has a moderate 50-splice site rather than the weak 50-splice site found in HIPK3-T. In contrast to the HIPK3-T exon, inactivation of one ESE in the SFRS10 exon 2 does not completely abolish Tra2b activation, likely due to the associated moderately strong 50-splice site. Hence the HIPK3-T and SFRS10 exons represent two prototypical examples of alternative exons regulated by the generally expressed splicing regu- lator protein Tra2b. Splicing inclusion can be either highly tissue-speciﬁc as for HIPK3-T or differentially regulated in various tissues as in SFRS10 exon 2. A general principle suggested from these two examples is that the anatomic spectrum of different Tra2b-responsive exons may be es- tablished in response to Tra2b concentration through an inter-play between the number of Tra2b-responsive ESEs and associated splice site strengths and other local splicing signals. Tra2b-responsive exons with stronger 50-splice sites might be activated more ubiquitously than weaker exons like HIPK3-T with weaker 50-splice sites. With these weaker exons, splicing inclusion would be switched on in speciﬁc tissues like the testis with elevated Tra2b concentrations and/or down-regulation of some splicing repressors (42). It is also possible that HIPK3-T is under regulation by trans-acting factors like PP1 which regulate Tra2b activity (43). We propose an array of Tra2b binding sites within the HIPK3-T exon are deciphered to give tissue speciﬁc patterns of splicing activation in response to the expres- sion levels of Tra2b. Very similar mechanisms of Tra2 splicing regulation have been shown to take place in Drosophila, both on the 30-splice site (44) and on 50-splice site regions (45). Activation of the Doublesex female-speciﬁc 30-splice site is modulated in response to the number of multi-site enhancers for Tra2, and activa- tion increases linearly rather than synergistically in response to this ESE number (17). In ﬂies, increases in numbers of Tra2-responsive ESEs have been suggested to increase the probability of assembly of a splicing acti- vator complex on Doublesex pre-mRNA, rather than enabling cooperative assembly of multiple binding splicing complexes containing Tra2. In this case the number of enhancer binding sites may inﬂuence the prob- ability of U1 snRNP binding to the alternatively spliced exon. However, despite potential subtle mechanistic dif- ferences, human and ﬂy orthologues of Tra2 protein seem to regulate splicing switches in a somewhat analogous fashion, both requiring assembly of Tra2 proteins on a responsive gauge. Regulated splice variants of other Drosophila mRNAs are similarly made in response to dif- ferent threshold values of Tra2 concentrations in different tissues (18). In this study we also found that Tra2b has a magniﬁed activating effect on intrinsically weak exons. This magniﬁed effect makes the HIPK3-T splicing switch more efﬁcient. Since the testis has an increased Tra2b con- centration (19), this observed splicing activation effect of Tra2b on weakly deﬁned exons is also likely to apply to other intrinsically weak exons with Tra2b binding sites, and thus might contribute to the high overall levels of exon inclusion observed in the testis (8–11). ACKNOWLEDGEMENTS The authors thank Dr Y. Durocher for the gift of the plasmid pTT3, and Dr F. Mauxion and B. Muller for