8098 Nucleic Acids Research, 2011, Vol. 39, No. 18 Figure 4. ESE-depleted versions of HIPK3-T are still able to bind Tra2b. (A) Gel shift experiments (Electrophoretic Mobility Shift Assays) were carried out with either wild-type HIPK3-T RNA, or HIPK3-T variants with different ESE proﬁles. Radiolabelled RNAs were shifted with 0, 50 and 100 ng added Tra2b protein. The sequences of these RNAs are given in Table 1. (B) Puriﬁed full length Tra2b Flag tagged protein. (C) RNA binding proteins associated with the HIPK3-T exon and downstream intronic region are associated with a number of RNA splicing regulators. wt = wild-type HIPK3-T exon, DS = downstream RNA. are absolutely required for Tra2b-mediated splicing acti- vation of HIPK3-T. Each of the identiﬁed ESEs within HIPK3-T contains at least one AGAA motif, consistent with the known speciﬁc binding site of Tra2b protein (15,16). However, after each single ESE disruption there were three AGAA-containing ESEs remaining, so we pre- dicted that such mutated HIPK3-T exons should still be able to bind to Tra2b protein even though splicing activa- tion was lost. To test this prediction we carried out gel shift experi- ments. In preliminary experiments we identiﬁed strong binding of bacterially expressed protein containing the RRM of Tra2b to the HIPK3-T exon and no binding to the downstream intronic RNA (data not shown). This indicated that the RRM was important for interaction with HIPK3-T in vitro. Since Tra2b protein can multimerize via its RS domains (31), full length Tra2b protein was then expressed and puriﬁed from mam- malian cells (Figure 4B). Puriﬁed protein was then used to test in vitro association with radiolabelled RNAs comprising either wild-type HIPK3-T, HIPK3-T exon with a single ESE disruption (B4), HIPK3-T exon with a double ESE disruption (B5), and HIPK3-T exon with a triple ESE disruption (B6). Strong and almost complete Tra2b binding was observed to wild-type HIPK3-T exon even at the lowest concentration of added Tra2b (Figure 4A, lanes 1–5 and 16–20). Additional band shifts were obtained with increasing concentrations of Tra2b, most likely due to binding of several Tra2b molecules to the T exon