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J. Biol. Chem., Vol. 280, Issue 26, 24532-24538, July 1, 2005

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Yeast mRNA Poly(A) Tail Length Control Can Be Reconstituted in Vitro in the Absence of Pab1p-dependent Poly(A) Nuclease Activity^*

Sonia Dheur¶, Keith R. Nykamp||**, Nicolas Viphakone, Maurice S. Swanson||, and Lionel Minvielle-Sebastia

From the CNRS UMR 5095, Institut de Biochimie et Génétique Cellulaires, 1 rue Camille Saint-Saëns, F-33077 Bordeaux cedex, France, the Université Victor Segalen Bordeaux 2, 146 rue Léo-Saignat, F-33076 Bordeaux cedex, France, and the ^||Department of Molecular Genetics and Microbiology, Powell Gene Therapy Center, University of Florida, College of Medicine, Gainesville, Florida 32610-0266

Regulation of poly(A) tail length during mRNA 3'-end formation requires a specific poly(A)-binding protein in addition to the cleavage/polyadenylation machinery. The mechanism that controls polyadenylation in mammals is well understood and involves the nuclear poly(A)-binding protein PABPN1. In contrast, poly(A) tail length regulation is poorly understood in yeast. Previous studies have suggested that the major cytoplasmic poly(A)-binding protein Pab1p acts as a length control factor in conjunction with the Pab1p-dependent poly(A) nuclease PAN, to regulate poly(A) tail length in an mRNA specific manner. In contrast, we recently showed that Nab2p regulates polyadenylation during de novo synthesis, and its nuclear location is more consistent with a role in 3'-end processing than that of cytoplasmic Pab1p. Here, we investigate whether PAN activity is required for de novo poly(A) tail synthesis. Components required for mRNA 3'-end formation were purified from wild-type and pan mutant cells. In both situations, 3'-end formation could be reconstituted whether Nab2p or Pab1p was used as the poly(A) tail length control factor. However, polyadenylation was more efficient and physiologically more relevant in the presence of Nab2p as opposed to Pab1p. Moreover, cell immunofluorescence studies confirmed that PAN subunits are localized in the cytoplasm which suggests that cytoplasmic Pab1p and PAN may act at a later stage in mRNA metabolism. Based on these findings, we propose that Nab2p is necessary and sufficient to regulate poly(A) tail length during de novo synthesis in yeast.

Received for publication, April 29, 2005

^* This work was supported by grants from the CNRS, the Ministère de la Recherche Scientifique, La Fondation pour la Recherche Médicale/Fondation BNP-Paribas, and La Ligue Nationale contre le Cancer (to L. M.-S.) and from the National Institutes of Health (to M. S. S.). 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.

^¶ In the course of this work, recipient of a postdoctoral long term fellowship from La Ligue Nationale contre le Cancer.

^** Present address: Howard Hughes Medical Inst., Dept. of Biochemistry, University of Wisconsin, Madison, WI 53706.

Supported by a predoctoral fellowship from the French Ministère de la Recherche et de la Technologie.

To whom correspondence should be addressed. Tel./Fax: 33-5-56-99-90-08; E-mail: lionel.minvielle{at}ibgc.u-bordeaux2.fr.

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