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Molecular and Cellular Biology, April 2009, p. 1834-1854, Vol. 29, No. 7
0270-7306/09/$08.00+0     doi:10.1128/MCB.01337-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

APE1/Ref-1 Interacts with NPM1 within Nucleoli and Plays a Role in the rRNA Quality Control Process ^,

Carlo Vascotto,^1, Damiano Fantini,^1, Milena Romanello,¹ Laura Cesaratto,¹ Marta Deganuto,¹ Antonio Leonardi,³ J. Pablo Radicella,⁴ Mark R. Kelley,^5,6,7 Chiara D'Ambrosio,² Andrea Scaloni,² Franco Quadrifoglio,¹ and Gianluca Tell^1*

Department of Biomedical Sciences and Technologies, University of Udine, 33100 Udine, Italy,¹ Proteomics and Mass Spectrometry Laboratory, ISPAAM, National Research Council, 80147 Naples, Italy,² Dipartimento di Biologia e Patologia Cellulare e Molecolare, Federico II University of Naples, 80131 Naples, Italy,³ CEA, Institut de Radiobiologie Cellulaire et Moléculaire, UMR217 CNRS, 92265 Fontenay-aux-Roses, France,⁴ Department of Pediatrics, Herman B Wells Center for Pediatric Research,⁵ Departments of Biochemistry and Molecular Biology,⁶ Pharmacology and Toxicology, Indiana University School of Medicine, 1044 W. Walnut, Indianapolis, Indiana⁷

Received 22 August 2008/ Returned for modification 29 September 2008/ Accepted 20 January 2009

APE1/Ref-1 (hereafter, APE1), a DNA repair enzyme and a transcriptional coactivator, is a vital protein in mammals. Its role in controlling cell growth and the molecular mechanisms that fine-tune its different cellular functions are still not known. By an unbiased proteomic approach, we have identified and characterized several novel APE1 partners which, unexpectedly, include a number of proteins involved in ribosome biogenesis and RNA processing. In particular, a novel interaction between nucleophosmin (NPM1) and APE1 was characterized. We observed that the 33 N-terminal residues of APE1 are required for stable interaction with the NPM1 oligomerization domain. As a consequence of the interaction with NPM1 and RNA, APE1 is localized within the nucleolus and this localization depends on cell cycle and active rRNA transcription. NPM1 stimulates APE1 endonuclease activity on abasic double-stranded DNA (dsDNA) but decreases APE1 endonuclease activity on abasic single-stranded RNA (ssRNA) by masking the N-terminal region of APE1 required for stable RNA binding. In APE1-knocked-down cells, pre-rRNA synthesis and rRNA processing were not affected but inability to remove 8-hydroxyguanine-containing rRNA upon oxidative stress, impaired translation, lower intracellular protein content, and decreased cell growth rate were found. Our data demonstrate that APE1 affects cell growth by directly acting on RNA quality control mechanisms, thus affecting gene expression through posttranscriptional mechanisms.

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* Corresponding author. Mailing address: Department of Biomedical Sciences and Technologies, University of Udine, P.le Kolbe 4, 33100 Udine, Italy. Phone: 39 0432 494311. Fax: 39 0432 494301. E-mail: gianluca.tell{at}uniud.it

Published ahead of print on 2 February 2009.

Supplemental material for this article may be found at http://mcb.asm.org/.

C.V. and D.F. contributed equally to the present work.

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Molecular and Cellular Biology, April 2009, p. 1834-1854, Vol. 29, No. 7
0270-7306/09/$08.00+0     doi:10.1128/MCB.01337-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.