Abrogation of DNA vector-based RNAi during apoptosis in mammalian cells due to caspase-mediated cleavage and inactivation of Dicer-1.
RNA interference (RNAi) is used as a reverse-genetic tool to examine functions of a gene in different cellular processes including apoptosis. As key cellular proteins are inactivated during apoptosis, and as RNAi requires cooperation of many cellular proteins, we examined whether DNA vector-based RNAi would continue to function during apoptosis. The short hairpin RNA transcribed from the DNA vector is processed by Dicer-1 to form small interfering RNA that is incorporated in the RNA-induced silencing complex (RISC) to guide a sequence-specific silencing of the target mRNA. We report here that DNA vector-based RNAi of three different genes, namely poly(ADP-ribose) polymerase-1, p14(ARF) and lamin A/C are abrogated during apoptosis. The failure of DNA vector-based RNAi was not at the level of Ago-2 or RISC-mediated step of RNAi but due to catalytic inactivation of Dicer-1 on specific cleavage at the STTD(1476) and CGVD(1538) sites within its RNase IIIa domain. Using multiple approaches, caspase-3 was identified as the major caspase responsible for the cleavage and inactivation of Dicer-1. As Dicer-1 is also the common endonuclease required for formation of microRNA (miRNA) in mammalian cells, we observed decreased levels of mature forms of miR-16, miR-21 and let-7a. Our results suggest a role for apoptotic cleavage and inactivation of Dicer-1 in controlling apoptotic events through altered availability of miRNA.
Ghodgaonkar
M M
MM
Laboratory for Skin Cancer Research, Faculty of Medicine, CHUL Research Center (CHUQ), Laval University, QC, Canada.
Shah
R G
RG
Kandan-Kulangara
F
F
Affar
E-B
EB
Qi
H H
HH
Wiemer
E
E
Shah
G M
GM
D000595:Amino Acid Sequence; D017209:Apoptosis; D053148:Caspase 3 / Q000378:metabolism*; D002460:Cell Line; D053487:DEAD-box RNA Helicases / Q000378:metabolism*; D005347:Fibroblasts / Q000378:metabolism; D055785:Gene Knockdown Techniques; D005822:Genetic Vectors / Q000235:genetics; D006367:HeLa Cells; D006801:Humans; D034904:Lamin Type A / Q000172:deficiency / Q000378:metabolism; D035683:MicroRNAs / Q000378:metabolism; D000071137:Poly (ADP-Ribose) Polymerase-1; D011065:Poly(ADP-ribose) Polymerases / Q000172:deficiency / Q000378:metabolism; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000378:metabolism; D034743:RNA-Induced Silencing Complex / Q000378:metabolism; D043244:Ribonuclease III / Q000378:metabolism*; D025803:Tumor Suppressor Protein p14ARF / Q000172:deficiency / Q000378:metabolism
C494847:LMNA protein, human; D034904:Lamin Type A; D035683:MicroRNAs; D034741:RNA, Small Interfering; D034743:RNA-Induced Silencing Complex; D025803:Tumor Suppressor Protein p14ARF; C499320:PARP1 protein, human; D000071137:Poly (ADP-Ribose) Polymerase-1; D011065:Poly(ADP-ribose) Polymerases; C408087:DICER1 protein, human; D043244:Ribonuclease III; D053148:Caspase 3; D053487:DEAD-box RNA Helicases