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Precursor microRNA-programmed silencing complex assembly pathways in mammals.
Publication Type:Journal Article
Source:Mol Cell, Volume 46, Issue 4, p.507-17 (2012)
Keywords:Animals, Argonaute Proteins, Base Sequence, Cell Line, DEAD-box RNA Helicases, Mice, Mice, Knockout, MicroRNAs, Models, Biological, Protein Multimerization, Ribonuclease III, RNA Processing, Post-Transcriptional, RNA-Induced Silencing Complex
<p>Assembly 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.</p>