Silencing of SPC2 expression using an engineered delta ribozyme in the mouse betaTC-3 endocrine cell line.
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 betaTC-3 stable cell lines expressing the chimeric tRNA(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 betaTC-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.
D'Anjou
François
F
Départements de Pharmacologie et de Biochimie, Université de Sherbrooke, Sherbrooke, Québec J1H 5N4, Canada.
Bergeron
Lucien Junior
LJ
Larbi
Nadia Ben
NB
Fournier
Isabelle
I
Salzet
Michel
M
Perreault
Jean-Pierre
JP
Day
Robert
R
D000818:Animals; D045744:Cell Line, Tumor; D006224:Cricetinae; D005818:Genetic Engineering; D003698:Hepatitis Delta Virus / Q000201:enzymology*; D007340:Insulinoma; D013058:Mass Spectrometry; D051379:Mice; D045706:Proprotein Convertase 2 / Q000235:genetics*; D034622:RNA Interference; D016337:RNA, Catalytic / Q000737:chemistry / Q000378:metabolism*; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D012366:RNA, Transfer, Val / Q000235:genetics; D013379:Substrate Specificity
D016337:RNA, Catalytic; D012333:RNA, Messenger; D012366:RNA, Transfer, Val; C473668:Pcsk2 protein, mouse; D045706:Proprotein Convertase 2