کد مقاله | کد نشریه | سال انتشار | مقاله انگلیسی | نسخه تمام متن |
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1996588 | 1065491 | 2012 | 11 صفحه PDF | دانلود رایگان |
SummaryAssembly of microRNA ribonucleoproteins (miRNPs) or RNA-induced silencing complexes (RISCs) is essential for the function of miRNAs and initiates from processing of precursor miRNAs (pre-miRNAs) by Dicer or by Ago2. Here, we report an in vitro miRNP/RISC assembly assay programmed by pre-miRNAs from mammalian cell lysates. Combining in vivo studies in Dicer Knockout cells reconstituted with wild-type or catalytically inactive Dicer, we find that the miRNA loading complex (miRLC) is the primary machinery linking pre-miRNA processing to miRNA loading. We show that a miRNA precursor deposit complex (miPDC) plays a crucial role in Dicer-independent miRNA biogenesis and promotes miRNP assembly of certain Dicer-dependent miRNAs. Furthermore, we find that 5′-uridine, 3′-mid base pairing, and 5′-mid mismatches within pre-miRNAs promote their assembly into miPDC. Our studies provide a comprehensive view of miRNP/RISC assembly pathways in mammals, and our assay provides a versatile platform for further mechanistic dissection of such pathways in mammals.
► Development of in vitro miRNP/RISC assembly assay programmed by pre-miRNAs
► miRLC is the primary machinery that links pre-miRNA processing to miRNA loading
► Roles of miPDC in miRNA biogenesis
► 5′-U, 3′-mid base pairs, 5′-mid mismatches in pre-miRNAs promote miPDC assembly
Journal: - Volume 46, Issue 4, 25 May 2012, Pages 507–517