title: Precursor microRNA-programmed silencing complex assembly pathways in mammals.
abstract: 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.
authors.affiliation: Department of Pathology and Laboratory Medicine, Division of Neuropathology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA.
authors.forename: Xuhang
authors.initials: X
authors.lastname: Liu
authors.forename: Dong-Yan
authors.initials: DY
authors.lastname: Jin
authors.forename: Michael T
authors.initials: MT
authors.lastname: McManus
authors.forename: Zissimos
authors.initials: Z
authors.lastname: Mourelatos
mesh_terms: D000818:Animals; D060565:Argonaute Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D001483:Base Sequence; D002460:Cell Line; D053487:DEAD-box RNA Helicases / Q000737:chemistry / Q000172:deficiency / Q000235:genetics / Q000378:metabolism; D066298:In Vitro Techniques; D051379:Mice; D018345:Mice, Knockout; D035683:MicroRNAs / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D008954:Models, Biological; D055503:Protein Multimerization; D012323:RNA Processing, Post-Transcriptional; D034743:RNA-Induced Silencing Complex / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D043244:Ribonuclease III / Q000737:chemistry / Q000172:deficiency / Q000235:genetics / Q000378:metabolism
chemical_list: C526735:Ago2 protein, mouse; D060565:Argonaute Proteins; D035683:MicroRNAs; D034743:RNA-Induced Silencing Complex; C509781:Dicer1 protein, mouse; D043244:Ribonuclease III; D053487:DEAD-box RNA Helicases