Molecular design of a splicing switch responsive to the RNA binding protein Tra2β

Tra2β regulates a number of splicing switches including activation of the human testis-specific exon HIPK3-T in the Homeodomain Interacting Protein Kinase 3 gene. By testing HIPK3-T exons of different intrinsic strengths, we found Tra2β most efficiently activated splicing inclusion of intrinsically weak exons, although these were spliced at a lower overall level. Both the RRM and N-terminal RS-rich region of Tra2β were required for splicing activation. Bioinformatic searches for splicing enhancers and repressors mapped four physically distinct exonic splicing enhancers (ESEs) within HIPK3-T, each containing the known Tra2β AGAA-rich binding site. Surprisingly disruption of each single ESE prevented Tra2β-mediated activation, although single mutated exons could still bind Tra2β protein by gel shifts and functional splicing analyses. Titration experiments indicate an additive model of HIPK3-T splicing activation, requiring availability of an array of four distinct ESEs to enable splicing activation. To enable this efficient Tra2β-mediated splicing switch to operate, a closely adjacent downstream and potentially competitive stronger 5'-splice site is actively repressed. Our data indicate that a novel arrangement of multiple mono-specific AGAA-rich ESEs coupled to a weak 5'-splice site functions as a responsive gauge. This gauge monitors changes in the specific nuclear concentration of the RNA binding protein Tra2β, and co-ordinately regulates HIPK3-T exon splicing inclusion.