title: A heterotrimer model of the complete Microprocessor complex revealed by single-molecule subunit counting.
abstract: During microRNA (miRNA) biogenesis, the Microprocessor complex (MC), composed minimally of Drosha, an RNaseIII enzyme, and DGCR8, a double-stranded RNA-binding protein, cleaves the primary-miRNA (pri-miRNA) to release the pre-miRNA stem-loop structure. Size-exclusion chromatography of the MC, isolated from mammalian cells, suggested multiple copies of one or both proteins in the complex. However, the exact stoichiometry was unknown. Initial experiments suggested that DGCR8 bound pri-miRNA substrates specifically, and given that Drosha could not be bound or cross-linked to RNA, a sequential model for binding was established in which DGCR8 bound first and recruited Drosha. Therefore, many laboratories have studied DGCR8 binding to RNA in the absence of Drosha and have shown that deletion constructs of DGCR8 can multimerize in the presence of RNA. More recently, it was demonstrated that Drosha can bind pri-miRNA substrates in the absence of DGCR8, casting doubt on the sequential model of binding. In the same study, using a single-molecule photobleaching assay, fluorescent protein-tagged deletion constructs of DGCR8 and Drosha assembled into a heterotrimeric complex on RNA, comprising two DGCR8 molecules and one Drosha molecule. To determine the stoichiometry of Drosha and DGCR8 within the MC in the absence of added RNA, we also used a single-molecule photobleaching assay and confirmed the heterotrimeric model of the human MC. We demonstrate that a heterotrimeric complex is likely preformed in the absence of RNA and exists even when full-length proteins are expressed and purified from human cells, and when hAGT-derived tags are used rather than fluorescent proteins.
authors.affiliation: Department of Molecular Biophysics and Biochemistry, Howard Hughes Medical Institute, Yale University School of Medicine, New Haven, Connecticut 06536, USA Department of Microbiology, Center for Scientific Research and Higher Education of Ensenada (CICESE), Ensenada, Baja California 22860, Mexico.
authors.forename: Kristina M
authors.initials: KM
authors.lastname: Herbert
authors.affiliation: Laboratory of Molecular Biophysics, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, Maryland 20892, USA.
authors.forename: Susanta K
authors.initials: SK
authors.lastname: Sarkar
authors.affiliation: Laboratory of Molecular Biophysics, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, Maryland 20892, USA.
authors.forename: Maria
authors.initials: M
authors.lastname: Mills
authors.affiliation: Department of Microbiology, Center for Scientific Research and Higher Education of Ensenada (CICESE), Ensenada, Baja California 22860, Mexico.
authors.forename: Hilda C
authors.initials: HC
authors.lastname: Delgado De la Herran
authors.affiliation: Laboratory of Molecular Biophysics, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, Maryland 20892, USA.
authors.forename: Keir C
authors.initials: KC
authors.lastname: Neuman
authors.affiliation: Department of Molecular Biophysics and Biochemistry, Howard Hughes Medical Institute, Yale University School of Medicine, New Haven, Connecticut 06536, USA.
authors.forename: Joan A
authors.initials: JA
authors.lastname: Steitz
mesh_terms: D000818:Animals; D036681:Fluorescence Recovery After Photobleaching; D005456:Fluorescent Dyes / Q000737:chemistry; D005786:Gene Expression Regulation; D057809:HEK293 Cells; D006801:Humans; D035683:MicroRNAs / Q000235:genetics / Q000378:metabolism*; D009690:Nucleic Acid Conformation; D010957:Plasmids / Q000737:chemistry; D011485:Protein Binding; D055503:Protein Multimerization; D016601:RNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D043244:Ribonuclease III / Q000235:genetics / Q000378:metabolism*; D015398:Signal Transduction; D013194:Staining and Labeling; D014162:Transfection
chemical_list: C474043:DGCR8 protein, human; D005456:Fluorescent Dyes; D035683:MicroRNAs; D016601:RNA-Binding Proteins; C517918:DROSHA protein, human; D043244:Ribonuclease III
keywords: Microprocessor complex; miRNA; single-molecule; stoichiometry