Precursor MicroRNA-Programmed Silencing Complex Assembly Pathways in Mammals

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