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J. Biol. Chem., Vol. 278, Issue 47, 46357-46368, November 21, 2003

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Identification of a C-terminal Poly(A)-binding Protein (PABP)-PABP Interaction Domain

ROLE IN COOPERATIVE BINDING TO POLY(A) AND EFFICIENT CAP DISTAL TRANSLATIONAL REPRESSION^*

Eduardo O. Melo, Rafael Dhalia¶, Cezar Martins de Sa, Nancy Standart||, and Osvaldo P. de Melo Neto¶**

From the Departamento de Biologia Celular, Universidade de Brasilia, Brasilia DF 70910-900, Brazil, Embrapa Recursos Genéticos e Biotecnologia, Parque Rural, Final W5, Asa Norte, Brasília DF 70770-900, Brazil, ^¶Centro de Pesquisas Aggeu Magalhães, Fundação Oswaldo Cruz, Campus da Universidade Federal de Pernambuco, Avenida Moraes Rego s/n, Recife PE 50670-420, Brazil, and the ^||Department of Biochemistry, University of Cambridge, Tennis Court Road, Cambridge CB2 1GA, United Kingdom

The poly(A)-binding protein (PABP), bound to the 3' poly(A) tail of eukaryotic mRNAs, plays critical roles in mRNA translation and stability. PABP autoregulates its synthesis by binding to a conserved A-rich sequence present in the 5'-untranslated region of PABP mRNA and repressing its translation. PABP is composed of two parts: the highly conserved N terminus, containing 4 RNA recognition motifs (RRMs) responsible for poly(A) and eIF4G binding; and the more variable C terminus, which includes the recently described PABC domain, and promotes intermolecular interaction between PABP molecules as well as cooperative binding to poly(A). Here we show that, in vitro, GST-PABP represses the translation of reporter mRNAs containing 20 or more A residues in their 5'-untranslated regions and remains effective as a repressor when an A₆₁ tract is placed at different distances from the cap, up to 126 nucleotides. Deletion of the PABP C terminus, but not the PABC domain alone, significantly reduces its ability to inhibit translation when bound to sequences distal to the cap, but not to proximal ones. Moreover, cooperative binding by multiple PABP molecules to poly(A) requires the C terminus, but not the PABC domain. Further analysis using pull-down assays shows that the interaction between PABP molecules, mediated by the C terminus, does not require the PABC domain and is enhanced by the presence of RRM 4. In vivo, fusion proteins containing parts of the PABP C terminus fused to the viral coat protein MS2 have an enhanced ability to prevent the expression of chloramphenicol acetyltransferase reporter mRNAs containing the MS2 binding site at distal distances from the cap. Altogether, our results identify a proline- and glutamine-rich linker located between the RRMs and the PABC domain as being strictly required for PABP/PABP interaction, cooperative binding to poly(A) and enhanced translational repression of reporter mRNAs in vitro and in vivo.

Received for publication, July 15, 2003 , and in revised form, August 25, 2003.

^* This work was supported by the Conselho Nacional de Desenvolvimento Cientifico e Tecnológico, Coordemação de Aperfeiçoamento de Pessoal de Nível Superior, and the British Council. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^** To whom correspondence should be addressed. Tel.: 55-81-3301-2568; Fax: 55-81-3453-2449; E-mail: opmn{at}cpqam.fiocruz.br.

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