Silencing of SPC2 Expression Using an Engineered δ Ribozyme in the Mouse βTC-3 Endocrine Cell Line

• Published in: The Journal of biological chemistry 2004-04, Vol.279 (14), p.14232-14239
• Main Authors: D'Anjou, François, Bergeron, Lucien Junior, Larbi, Nadia Ben, Fournier, Isabelle, Salzet, Michel, Perreault, Jean-Pierre, Day, Robert
• Format: Article
• Language: English
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M310632200

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 delta 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 beta TC-3 stable cell lines expressing the chimeric tRNA super(Val)- delta 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 beta 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.