A forward genetic screen to study mammalian RNA interference: essential role of RNase IIIa domain of Dicer1 in 3' strand cleavage of dsRNA in vivo.
RNA interference is a major post-transcriptional regulatory pathway in many eukaryotes. The RNase III enzyme Dicer1 processes precursor RNAs into small RNA duplexes to be loaded onto Argonaute proteins, the effector components of RNA-induced silencing complex. Biochemical studies have shown that the RNase IIIa and RNase IIIb domains of Dicer1 cleave the 3' and 5' strands of dsRNAs, respectively, although the in vivo functional significance of this activity remains unclear. Genetic screening of mammalian cells is useful for studying molecular mechanisms at the cellular level. In the present study, we conducted a novel forward genetic screen for mammalian RNA interference components using Chinese hamster ovary cells and successfully obtained several Dicer1 mutant lines. One mutant bore an intriguing Dicer1 allele in which a conserved glutamic acid in the RNase IIIa domain was substituted with a lysine. Our detailed cell biological study demonstrated that the RNase IIIa domain of Dicer1 was essential for generating small RNAs embedded in the 3' stem of exogenous hairpin-like RNAs. In the mutant cells, the expression of endogenous mature microRNAs derived from the 3' stem of pre-microRNA was repressed more severely than that from the 5' stem. Moreover, appropriate processing and loading of small RNAs were required for the dissociation of Argonaute 2 from Dicer1. The data obtained in the present study demonstrate that this screening method represents a promising strategy for the identification of unknown components of mammalian RNA interference pathways and the study of the biological significance of these components at the cellular level.
Department of Pathology, Medical School, Osaka University, Japan.
Kazuhito
K
Ohishi
Toru
T
Nakano
D000818:Animals; D060565:Argonaute Proteins / Q000235:genetics / Q000378:metabolism; D016466:CHO Cells; D006224:Cricetinae; D003412:Cricetulus; D053487:DEAD-box RNA Helicases; D005786:Gene Expression Regulation; D005820:Genetic Testing; D018698:Glutamic Acid / Q000235:genetics / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D008156:Luciferases / Q000378:metabolism; D035683:MicroRNAs / Q000235:genetics; D016297:Mutagenesis, Site-Directed; D009154:Mutation / Q000235:genetics; D017434:Protein Structure, Tertiary; D034622:RNA Interference; D012323:RNA Processing, Post-Transcriptional; D012330:RNA, Double-Stranded / Q000378:metabolism*; D012333:RNA, Messenger / Q000235:genetics; D034741:RNA, Small Interfering / Q000235:genetics*; D034743:RNA-Induced Silencing Complex; D060888:Real-Time Polymerase Chain Reaction; D043244:Ribonuclease III / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*
D060565:Argonaute Proteins; D035683:MicroRNAs; D012330:RNA, Double-Stranded; D012333:RNA, Messenger; D034741:RNA, Small Interfering; D034743:RNA-Induced Silencing Complex; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; D018698:Glutamic Acid; D008156:Luciferases; C408087:DICER1 protein, human; D043244:Ribonuclease III; D053487:DEAD-box RNA Helicases