title: Characterization of the miRNA-RISC loading complex and miRNA-RISC formed in the Drosophila miRNA pathway.
abstract: In Drosophila, miRNA is processed by Dicer-1 (DCR-1) from its precursor and loaded onto Argonaute1 (AGO1). AGO1 recognizes target mRNAs based on the miRNA sequence and suppresses the expression at post-transcriptional levels. GW182, a P-body component, localizes the AGO1 complex to processing bodies (P-bodies) where mRNA targets are decayed or stored. However, the details of the pathway remain elusive. In this study, two distinct types of AGO1-containing complexes from Drosophila Schneider2 (S2) cells were isolated and compared at the molecular level. The AGO1 complex with DCR-1 contained neither mature miRNA nor GW182 but exhibited pre-miRNA processing activity. The resultant mature RNA was loaded onto AGO1 within the complex. The AGO1 complex with GW182 excluded DCR-1, but possessed mature miRNA and showed no pre-miRNA processing activity. Thus, the AGO1-DCR-1 and AGO1-GW182 complexes correspond to miRLC (miRISC loading complex) and miRISC, respectively. The requirement for various domains of AGO1 in miRNA-loading and DCR-1/GW182 interaction was also examined. The Mid domain mutant (F2V2) interacted with DCR-1 but not with mature miRNA and GW182. The AGO1-PAZ mutant lacks the mature miRNA-binding ability but associates with either DCR-1 or GW182. The AGO1-PIWI mutant showed no Slicer activity but associates with mature miRNA. These results indicate that these domains are required differently for miRLC and miRISC formation in the miRNA pathway.
authors.affiliation: School of Medicine, Keio University, Tokyo 160-8582, Japan.
authors.forename: Keita
authors.initials: K
authors.lastname: Miyoshi
authors.forename: Tomoko N
authors.initials: TN
authors.lastname: Okada
authors.forename: Haruhiko
authors.initials: H
authors.lastname: Siomi
authors.forename: Mikiko C
authors.initials: MC
authors.lastname: Siomi
mesh_terms: D000818:Animals; D060565:Argonaute Proteins; D015152:Blotting, Northern; D015153:Blotting, Western; D029721:Drosophila Proteins / Q000235:genetics / Q000378:metabolism; D004331:Drosophila melanogaster / Q000235:genetics* / Q000254:growth & development / Q000378:metabolism*; D039642:Eukaryotic Initiation Factors; D005455:Fluorescent Antibody Technique; D047468:Immunoprecipitation; D035683:MicroRNAs / Q000235:genetics / Q000378:metabolism*; D020365:RNA Helicases / Q000235:genetics / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism*; D034741:RNA, Small Interfering / Q000494:pharmacology; D034743:RNA-Induced Silencing Complex / Q000235:genetics / Q000378:metabolism*; D043244:Ribonuclease III / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction
chemical_list: C489654:AGO1 protein, Drosophila; D060565:Argonaute Proteins; D029721:Drosophila Proteins; D039642:Eukaryotic Initiation Factors; D035683:MicroRNAs; D012333:RNA, Messenger; D034741:RNA, Small Interfering; D034743:RNA-Induced Silencing Complex; C515033:Dcr-1 protein, Drosophila; D043244:Ribonuclease III; D020365:RNA Helicases