Characterization of the miRNA-RISC loading complex and miRNA-RISC formed in the Drosophila miRNA pathway.
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.
Miyoshi
Keita
K
School of Medicine, Keio University, Tokyo 160-8582, Japan.
Okada
Tomoko N
TN
Siomi
Haruhiko
H
Siomi
Mikiko C
MC
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
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