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

Grellscheid, S.N. and Dalgliesh, C. and Rozanska, A. and Grellscheid, D. and Bourgeois, C.F. and Stévenin, J. and Elliott, D.J. (2011) 'Molecular design of a splicing switch responsive to the RNA binding protein Tra2β.', Nucleic acids research., 39 (18). pp. 8092-8104.

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.

Item Type:Article
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Status:Peer-reviewed
Publisher Web site:http://dx.doi.org/10.1093/nar/gkr495
Publisher statement:© 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.
Record Created:15 Feb 2013 10:05
Last Modified:15 Feb 2013 10:52

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