PTB (polypyrimidine tract-binding protein) is an abundant and widely expressed RNA-binding protein with four RRM (RNA recognition motif) domains. PTB is involved in numerous post-transcriptional steps in gene expression in both the nucleus and cytoplasm, but has been best characterized as a regulatory repressor of some ASEs (alternative splicing events), and as an activator of translation driven by IRESs (internal ribosome entry segments). We have used a variety of approaches to characterize the activities of PTB and its molecular interactions with RNA substrates and protein partners. Using splice-sensitive microarrays we found that PTB acts not only as a splicing repressor but also as an activator, and that these two activities are determined by the location at which PTB binds relative to target exons. We have identified minimal splicing repressor and activator domains, and have determined high resolution structures of the second RRM domain of PTB binding to peptide motifs from the co-repressor protein Raver1. Using single-molecule techniques we have determined the stoichiometry of PTB binding to a regulated splicing substrate in whole nuclear extracts. Finally, we have used tethered hydroxyl radical probing to determine the locations on viral IRESs at which each of the four RRM domains bind. We are now combining tethered probing with single molecule analyses to gain a detailed understanding of how PTB interacts with pre-mRNA substrates to effect either repression or activation of splicing.
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August 2012
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Conference Article|
July 20 2012
Defining the roles and interactions of PTB
Panagiota Kafasla;
Panagiota Kafasla
1
*Department of Biochemistry, University of Cambridge, Tennis Court Road, Cambridge CB2 1QW, U.K.
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Ian Mickleburgh;
Ian Mickleburgh
*Department of Biochemistry, University of Cambridge, Tennis Court Road, Cambridge CB2 1QW, U.K.
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Miriam Llorian;
Miriam Llorian
*Department of Biochemistry, University of Cambridge, Tennis Court Road, Cambridge CB2 1QW, U.K.
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Miguel Coelho;
Miguel Coelho
*Department of Biochemistry, University of Cambridge, Tennis Court Road, Cambridge CB2 1QW, U.K.
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Clare Gooding;
Clare Gooding
*Department of Biochemistry, University of Cambridge, Tennis Court Road, Cambridge CB2 1QW, U.K.
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Dmitry Cherny;
Dmitry Cherny
†Department of Biochemistry, University of Leicester, Leicester LE1 9HN, U.K.
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Amar Joshi;
Amar Joshi
2
‡Division of Cell and Molecular Biology, Imperial College London, Exhibition Road, London SW7 2AZ, U.K.
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Olga Kotik-Kogan;
Olga Kotik-Kogan
‡Division of Cell and Molecular Biology, Imperial College London, Exhibition Road, London SW7 2AZ, U.K.
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Stephen Curry;
Stephen Curry
‡Division of Cell and Molecular Biology, Imperial College London, Exhibition Road, London SW7 2AZ, U.K.
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Ian C. Eperon;
Ian C. Eperon
†Department of Biochemistry, University of Leicester, Leicester LE1 9HN, U.K.
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Richard J. Jackson;
Richard J. Jackson
*Department of Biochemistry, University of Cambridge, Tennis Court Road, Cambridge CB2 1QW, U.K.
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Christopher W.J. Smith
Christopher W.J. Smith
3
*Department of Biochemistry, University of Cambridge, Tennis Court Road, Cambridge CB2 1QW, U.K.
3To whom correspondence should be addressed (emailcwjs1@cam.ac.uk).
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Publisher: Portland Press Ltd
Received:
February 13 2012
Online ISSN: 1470-8752
Print ISSN: 0300-5127
© The Authors Journal compilation © 2012 Biochemical Society
2012
Biochem Soc Trans (2012) 40 (4): 815–820.
Article history
Received:
February 13 2012
Citation
Panagiota Kafasla, Ian Mickleburgh, Miriam Llorian, Miguel Coelho, Clare Gooding, Dmitry Cherny, Amar Joshi, Olga Kotik-Kogan, Stephen Curry, Ian C. Eperon, Richard J. Jackson, Christopher W.J. Smith; Defining the roles and interactions of PTB. Biochem Soc Trans 1 August 2012; 40 (4): 815–820. doi: https://doi.org/10.1042/BST20120044
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