title: Molecular design of a splicing switch responsive to the RNA binding protein Tra2β.
abstract: 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.
authors: Institute of Genetic Medicine, Newcastle University, Newcastle upon Tyne, NE1 3BZ, UK.
authors: Sushma Nagaraja
authors: SN
authors: Grellscheid
authors: Caroline
authors: C
authors: Dalgliesh
authors: Agata
authors: A
authors: Rozanska
authors: David
authors: D
authors: Grellscheid
authors: Cyril F
authors: CF
authors: Bourgeois
authors: James
authors: J
authors: Stévenin
authors: David J
authors: DJ
authors: Elliott
mesh_terms: D017398:Alternative Splicing; D005091:Exons; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins / Q000235:genetics* / Q000378:metabolism; D009419:Nerve Tissue Proteins / Q000737:chemistry / Q000378:metabolism*; D017346:Protein Serine-Threonine Kinases / Q000235:genetics* / Q000378:metabolism; D017434:Protein Structure, Tertiary; D022821:RNA Splice Sites; D016601:RNA-Binding Proteins / Q000737:chemistry / Q000378:metabolism*; D038621:Regulatory Sequences, Ribonucleic Acid; D000068103:Serine-Arginine Splicing Factors
chemical_list: D047908:Intracellular Signaling Peptides and Proteins; D009419:Nerve Tissue Proteins; D022821:RNA Splice Sites; D016601:RNA-Binding Proteins; D038621:Regulatory Sequences, Ribonucleic Acid; C097083:TRA2B protein, human; D000068103:Serine-Arginine Splicing Factors; C109102:HIPK3 protein, human; D017346:Protein Serine-Threonine Kinases