Persistence of different forms of transient RNAi during apoptosis in mammalian cells.
Gene silencing by transient or stable RNA-interference (RNAi) is used for the study of apoptosis with an assumption that apoptotic events will not influence RNAi. However, we recently reported that stable RNAi, i.e., a permanent gene-knockdown mediated by shRNA-generating DNA vectors that are integrated in the genome, fails rapidly after induction of apoptosis due to caspase-3-mediated cleavage and inactivation of the endoribonuclease Dicer-1 that is required for conversion of shRNA to siRNA. Since apoptosis studies also increasingly employ transient RNAi models in which apoptosis is induced immediately after a gene is temporarily knocked down within a few days of transfection with RNAi-inducing agents, we examined the impact of apoptosis on various models of transient RNAi. We report here that unlike the stable RNAi, all forms of transient RNAi, whether Dicer-1-independent (by 21mer dsRNA) or Dicer-1-dependent (by 27mer dsRNA or shRNA-generating DNA vector), whether for an exogenous gene GFP or an endogenous gene poly(ADP-ribose) polymerase-1, do not fail for 2-3 days after onset of apoptosis. Our results reflect the differences in dynamics of achieving and maintaining RNAi during the early phase after transfection in the transient RNAi model and the late steady-state phase of gene-knockdown in stable RNAi model. Our results also sound a cautionary note that RNAi status should be frequently validated in the studies involving apoptosis and that while stable RNAi can be safely used for the study of early apoptotic events, transient RNAi is more suitable for the study of both early and late apoptotic events.
Kandan-Kulangara
Febitha
F
Laboratory for Skin Cancer Research, Department of Molecular Biology, Medical Biochemistry and Pathology, Faculty of Medicine, Hospital Research Centre of Laval University (CHUL/CHUQ), Laval University, Quebec, Quebec, Canada.
Shah
Rashmi G
RG
Affar
El Bachir
el B
Shah
Girish M
GM
D000818:Animals; D017209:Apoptosis / Q000235:genetics; D016466:CHO Cells; D006224:Cricetinae; D003412:Cricetulus; D053487:DEAD-box RNA Helicases / Q000378:metabolism; D055785:Gene Knockdown Techniques; D049452:Green Fluorescent Proteins / Q000172:deficiency / Q000235:genetics; D006801:Humans; D011065:Poly(ADP-ribose) Polymerases / Q000172:deficiency / Q000235:genetics; D034622:RNA Interference; D043244:Ribonuclease III / Q000378:metabolism; D013997:Time Factors
D049452:Green Fluorescent Proteins; D011065:Poly(ADP-ribose) Polymerases; C408087:DICER1 protein, human; D043244:Ribonuclease III; D053487:DEAD-box RNA Helicases
Biochem Biophys Res Commun. 2005 May 27;331(1):167-74
15845374
J Biol Chem. 2008 May 23;283(21):14242-7
18375381
Trends Genet. 2003 Jan;19(1):9-12
12493242
Cancer Lett. 2006 Nov 8;243(1):38-46
16387423
Cell. 2006 Jul 28;126(2):231-5
16873051
Angew Chem Int Ed Engl. 2009;48(8):1378-98
19153977
Cell Death Differ. 2009 Jun;16(6):858-68
19229243
Nat Biotechnol. 2005 Feb;23(2):222-6
15619617
Nucleic Acids Res. 2009 Apr;37(7):e56
19282452