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Molecular and Cellular Biology, May 2001, p. 3364-3374, Vol. 21, No. 10
0270-7306/01/$04.00+0   DOI: 10.1128/MCB.21.10.3364-3374.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.

Protein Factor Requirements of the Apaf-1 Internal Ribosome Entry Segment: Roles of Polypyrimidine Tract Binding Protein and upstream of N-ras

Sally A. Mitchell,1 Emma C. Brown,2 Mark J. Coldwell,1 Richard J. Jackson,2 and Anne E. Willis1,*

Department of Biochemistry, University of Leicester, Leicester LE1 7RH,1 and Department of Biochemistry, University of Cambridge, Cambridge CB2 1GA,2 United Kingdom

Received 14 December 2000/Returned for modification 26 January 2001/Accepted 15 February 2001

It has been reported previously that the 5' untranslated region of the mRNA encoding Apaf-1 (apoptotic protease-activating factor 1) has an internal ribosome entry site (IRES), whose activity varies widely among different cell types. Here it is shown that the Apaf-1 IRES is active in rabbit reticulocyte lysates, provided that the system is supplemented with polypyrimidine tract binding protein (PTB) and upstream of N-ras (unr), two cellular RNA binding proteins previously identified to be required for rhinovirus IRES activity. In UV cross-linking assays and electrophoretic mobility shift assays with individual recombinant proteins, the Apaf-1 IRES binds unr but not PTB; however, PTB binding occurs if unr is present. Over a range of different cell types there is a broad correlation between the activity of the Apaf-1 IRES and their content of PTB and unr. In cell lines deficient in these proteins, overexpression of PTB and unr stimulated Apaf-1 IRES function. This is the first example where an IRES in a cellular mRNA has been shown to be functionally dependent, both in vitro and in vivo, on specific cellular RNA binding proteins. Given the critical role of Apaf-1 in apoptosis, these results have important implications for the control of the apoptotic cascade.

* Corresponding author. Mailing address: Department of Biochemistry, University of Leicester, University Rd., Leicester LE1 7RH, United Kingdom. Fax: 0116-2523369. E-mail: aew5{at}le.ac.uk.

Molecular and Cellular Biology, May 2001, p. 3364-3374, Vol. 21, No. 10
0270-7306/01/$04.00+0   DOI: 10.1128/MCB.21.10.3364-3374.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.


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