Precursor MicroRNA-Programmed Silencing Complex Assembly Pathways in Mammals

• Published in: Molecular cell 2012-05, Vol.46 (4), p.507-517
• Main Authors: Liu, Xuhang, Jin, Dong-Yan, McManus, Michael T., Mourelatos, Zissimos
• Format: Article
• Language: English
• Subjects: Animals; Argonaute 2 protein; Argonaute Proteins - chemistry; Argonaute Proteins - genetics; Argonaute Proteins - metabolism; Base Sequence; biogenesis; Cell Line; DEAD-box RNA Helicases - chemistry; DEAD-box RNA Helicases - deficiency; DEAD-box RNA Helicases - genetics; DEAD-box RNA Helicases - metabolism; In Vitro Techniques; in vivo studies; mammals; Mice; Mice, Knockout; microRNA; MicroRNAs - chemistry; MicroRNAs - genetics; MicroRNAs - metabolism; Models, Biological; Protein Multimerization; Ribonuclease III - chemistry; Ribonuclease III - deficiency; Ribonuclease III - genetics; Ribonuclease III - metabolism; ribonucleoproteins; RNA Processing, Post-Transcriptional; RNA-Induced Silencing Complex - chemistry; RNA-Induced Silencing Complex - genetics; RNA-Induced Silencing Complex - metabolism
• ISSN: 1097-2765; 1097-4164
• DOI: 10.1016/j.molcel.2012.03.010

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. ► 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