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J. Biol. Chem., Vol. 279, Issue 14, 14232-14239, April 2, 2004

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Silencing of SPC2 Expression Using an Engineered Ribozyme in the Mouse TC-3 Endocrine Cell Line^*

François D'Anjou¶, Lucien Junior Bergeron||**, Nadia Ben Larbi, Isabelle Fournier, Michel Salzet, Jean-Pierre Perreault||, and Robert Day¶¶

From the Départements de Pharmacologie et de ^||Biochimie, Université de Sherbrooke, Sherbrooke, Québec J1H 5N4, Canada and Laboratoire de Neuroimmunologie des Annélides, UMR CNRS 8017, Université des Sciences et Technologies de Lille, 59650 Villeneuve d'Ascq, France

Endoproteolytic processing is carried out by subtilase-like pro-protein convertases in mammalian cells. In order to understand the distinct roles of a member of this family (SPC2), gene silencing in cultured cells is an ideal approach. Previous studies showed limited success in either the degree of inhibition obtained or the stability of the cell lines. Here we demonstrate the high potential of ribozyme as a post-transcriptional gene silencing tool in cultured cells. We used an expression vector based on the RNA polymerase III promoter to establish TC-3 stable cell lines expressing the chimeric tRNA^Val- ribozyme transcript targeting SPC2 mRNA. Northern and Western blot hybridizations showed a specific reduction of SPC2 mRNA and protein. Validation of processing effects was tested by measuring the levels of dynorphin A-(1-8), which are present in TC-3 cells as a result of the unique cleavage of dynorphin A-(1-17) by SPC2. Moreover, a differential proteomic analysis confirmed these results and allowed identification of secretogranin II as a potential substrate of SPC2. The development of efficient, specific, and durable silencing tools, such as described in the present work, will be of great importance in elucidating the functions of the subtilase-like pro-protein convertases in regard to peptide processing and derived cellular events.

Received for publication, September 25, 2003 , and in revised form, January 15, 2004.

^* This work was supported in part by grants from the Canadian Institutes of Health Research (to J.-P. P. and R. D.), Health Canada (to J.-P. P.), the CNRS (to I. F. and M. S.), the Ministere de l'Éducation Nationale, de la Recherche et des Technologies (to I. F. and M. S.), and the Fondation pour la Recherche Médicale (to I. F.). 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.

Both authors contributed equally to this work.

^¶ Recipient of a pre-doctoral fellowship from the Natural Sciences and Engineering Research Council of Canada.

^** Recipient of a pre-doctoral fellowship from the Canadian Institutes of Health Research.

Investigator from the Canadian Institutes of Health Research. To whom correspondence may be addressed: Dépt. de Biochimie, RNA Group, Université de Sherbrooke, 3001 12^e Ave. Nord, Sherbrooke, Québec J1H 5N4, Canada. Tel.: 819-564-5310; Fax: 819-564-5340; E-mail: jean-pierre.perreault{at}usherbrooke.ca.

^¶¶ Senior scholar of the Fonds de la Recherche en Santé du Québec. To whom correspondence may be addressed: Dépt. de Pharmacologie, Institut de Pharmacologie de Sherbrooke Université de Sherbrooke, 3001 12^e Ave. Nord, Sherbrooke, Québec J1H 5N4, Canada. Tel.: 819-564-5428; Fax: 819-564-5400; E-mail: robert.day{at}usherbrooke.ca.

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