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J. Biol. Chem., Vol. 283, Issue 13, 8266-8273, March 28, 2008

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Functional Genomic Characterization of mRNAs Associated with TcPUF6, a Pumilio-like Protein from Trypanosoma cruzi^*

Bruno Dallagiovanna, Alejandro Correa, Christian M. Probst, Fabiola Holetz, Pablo Smircich^¶, Alessandra Melo de Aguiar, Fernanda Mansur, Claudio Vieira da Silva^||, Renato A. Mortara^||, Beatriz Garat^¶, Gregory A. Buck^**, Samuel Goldenberg, and Marco A. Krieger¹

From the Instituto de Biologia Molecular do Paraná, Rua Professor Algacyr Munhoz Mader 3775, Curitiba 81350-010 PR, Brazil, the Fundação Oswaldo Cruz, Avenida Brasil 4365, Rio de Janeiro 21040-900 RJ, Brazil, the ^¶Laboratório de Interacciones Moleculares, Facultad de Ciencias, Iguá 4225, 11400 Montevideo, Uruguay, the ^||Departamento de Microbiologia, Imunologia e Parasitologia, Rua Botucatu 862-8a, UNIFESP, São Paulo 04023-062 SP, Brazil, and the ^**Center for the Study of Biological Complexity, Virginia Commonwealth University, Richmond, Virginia 23284

Trypanosoma cruzi is the protozoan parasite that causes Chagas disease or American trypanosomiasis. Kinetoplastid parasites could be considered as model organisms for studying factors involved in posttranscriptional regulation because they control gene expression almost exclusively at this level. The PUF (Pumilio/FBF1) protein family regulates mRNA stability and translation in eukaryotes, and several members have been identified in trypanosomatids. We used a ribonomic approach to identify the putative target mRNAs associated with TcPUF6, a member of the T. cruzi PUF family. TcPUF6 is expressed in discrete sites in the cytoplasm at various stages of the parasite life cycle and is not associated with the translation machinery. The overexpression of a tandem affinity purification-tagged TcPUF6 protein allowed the identification of associated mRNAs by affinity purification assays and microarray hybridization yielding nine putative target mRNAs. Whole expression analysis of transfected parasites showed that the mRNAs associated with TcPUF6 were down-regulated in populations overexpressing TcPUF6. The association of TcPUF6 with the TcDhh1 helicase in vivo and the cellular co-localization of these proteins in epimastigote forms suggest that TcPUF6 promotes degradation of its associated mRNAs through interaction with RNA degradation complexes. Analysis of the mRNA levels of the putative TcPUF6-regulated genes during the parasite life cycle showed that their transcripts were up-regulated in metacyclic trypomastigotes. In these infective forms no co-localization between TcPUF6 and TcDhh1 was observed. Our results suggest that TcPUF6 regulates the half-lives of its associated transcripts via differential association with mRNA degradation complexes throughout its life cycle.

Received for publication, April 12, 2007 , and in revised form, December 4, 2007.

^* This work was supported by Fundação Oswaldo Cruz, Programa de Núcleos de Excelência (Fundação Araucária), Conselho Nacional de Desenvolvimento Cientifico e Tecnológico (Conselho Nacional de Desenvolvimento Cientifico e Technologico (CNPq), PROSUL), and National Institutes of Health Grant 5R01AI050196-02. This work was also supported by CNPq research fellowships (to B. D., A. C., C. M. P., M. P. M., S. G., and M. A. K.) and a Programa de Estudio y Desarrollo de Ciencias Básicas fellowship and an AMSUD/Pasteur training fellowship (to P. 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.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Tables S1 and S2 and supplemental Figs. S1–S4.

¹ To whom correspondence should be addressed: Rua Professor Algacyr Munhoz Mader 3775, Curitiba 81350-010 PR, Brazil. Tel.: 5541-3316-3232; Fax: 5541-3316-3267; E-mail: mkrieger{at}tecpar.br.

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