8092–8104 Nucleic Acids Research, 2011, Vol. 39, No. 18 doi:10.1093/nar/gkr495 Published online 30 June 2011 Molecular design of a splicing switch responsive to the RNA binding protein Tra2b Sushma Nagaraja Grellscheid1, Caroline Dalgliesh 1 , Agata Rozanska 1 , David Grellscheid2, Cyril F. Bourgeois3, James Ste´ venin3 and David J. Elliott1,* 1Institute of Genetic Medicine, Newcastle University, Newcastle upon Tyne, NE1 3BZ, 2Department of Physics, Institute for Particle Physics Phenomenology, Durham University, DH1 3LE, UK and 3Institut de Ge´ ne´ tique et de Biologie Mole´ culaire et Cellulaire (IGBMC), Institut National de Sante´ et de Recherche Me´ dicale (INSERM) U964/Centre National de Recherche Scientifique (CNRS) UMR 7104/Universite´ de Strasbourg, 67404 Illkirch, France Received December 15, 2010; Revised May 27, 2011; Accepted May 29, 2011 ABSTRACT Tra2b 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 Tra2b most efficiently activated splicing inclusion of intrin- sically weak exons, although these were spliced at a lower overall level. Both the RRM and N-terminal RS-rich region of Tra2b 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 Tra2b AGAA-rich binding site. Surprisingly disruption of each single ESE prevented Tra2b-mediated activa- tion, although single mutated exons could still bind Tra2b 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 Tra2b-mediated splicing switch to operate, a closely adjacent downstream and potentially com- petitive stronger 50-splice site is actively repressed. Our data indicate that a novel arrangement of multiple mono-specific AGAA-rich ESEs coupled to a weak 50-splice site functions as a responsive gauge. This gauge monitors changes in the specific nuclear concentration of the RNA binding protein Tra2b, and co-ordinately regulates HIPK3-T exon splicing inclusion. INTRODUCTION In excess of 90% of human genes encode more than one mRNA isoform, with alternative splicing making an im- portant contribution (1–3). Alternative splicing patterns of pre-mRNAs can be modulated in response to the concen- tration of speciﬁc nuclear RNA binding proteins within particular cells and tissues (4,5). The biological effect of splicing regulators is mediated through interpretation of an intrinsic splicing code embedded within pre-mRNA se- quences. Individual splicing factors can both activate and repress exon inclusion, depending on where they bind in target pre-mRNAs (6,7). Although alternative splicing is found in every tissue, particularly high levels have been observed in the human brain and testes (8–11). The target sites for RNA binding proteins which make up the splicing code are being deciphered by experiments and bioinfor- matics (12), and must explain why some exons are spliced with wide anatomic distributions, while others have much more restricted tissue-speciﬁc patterns of splicing inclusion. The SR family are an important class of splicing regu- lator proteins which contain domains enriched in arginine and serine residues (RS domains). Classical SR protein splicing regulators typically have one or two RNA recog- nition motifs (RRMs) required for binding to their target pre-mRNAs, and a C-terminal RS domain (13,14). The SR-like splicing regulator protein Tra2 is conserved between fruit ﬂies and humans. Tra2 has an unusual modular structure compared with ‘classical’ SR proteins, with a central RRM ﬂanked on the N-terminal side by a protein sequence enriched in arginine–serine (RS) dipep- tides (19 RS dipeptides in humans), and on the C-terminal side by a hinge region and a smaller RS-rich region (seven RS dipeptides in humans). Tra2b protein binds most strongly to an AGAA target RNA sequence (15,16). *To whom correspondence should be addressed. Tel: +0191 241 8694; Fax: +0191 241 8666; Email: david.elliott@ncl.ac.uk Present address: Cyril F. Bourgeois, Institute of Molecular Biology and Pathology of CNR, Department of Biology and Biotechnology, University of Rome ‘Sapienza’, Rome, Italy The Author(s) 2011. Published by Oxford University Press. This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/ by-nc/3.0), which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.