title: Identification and characterization of protein interactions in the mammalian mRNA processing body using a novel two-hybrid assay.
abstract: Components of the mRNA processing body (P-body) regulate critical steps in mRNA storage, transport, translation and degradation. At the core of the P-body is the decapping complex, which removes the 5' cap from de-adenylated mRNAs and mediates an irreversible step in mRNA degradation. The assembly of P-bodies in Saccharomyces cerevisiae, Arabidopsis thaliana and Drosophila melanogaster has been previously described. Less is known about the assembly of mammalian P-bodies. To investigate the interactions that occur between components of mammalian P-bodies, we developed a fluorescence-based, two-hybrid assay system. The assay depends on the ability of one P-body component, fused to an exogenous nuclear localization sequence (NLS), to recruit other P-body components to the nucleus. The assay was used to investigate interactions between P-body components Ge-1, DCP2, DCP1, EDC3, RAP55, and RCK. The results of this study show that the modified two-hybrid assay can be used to identify protein interactions that occur in a macromolecular complex. The assay can also be used to efficiently detect protein interaction domains. The results provide important insights into mammalian P-body assembly and demonstrate similarities, and critical differences, between P-body assembly in mammalian cells compared with that of other species.
authors.affiliation: Department of Medicine, Harvard Medical School, Center for Immunology and Inflammatory Diseases of the General Medical Services, Massachusetts General Hospital, 55 Fruit Street, Boston, MA 02114, USA. bloch@helix.mgh.harvard.edu
authors.forename: Donald B
authors.initials: DB
authors.lastname: Bloch
authors.forename: Rita A
authors.initials: RA
authors.lastname: Nobre
authors.forename: Gillian A
authors.initials: GA
authors.lastname: Bernstein
authors.forename: Wei-Hong
authors.initials: WH
authors.lastname: Yang
mesh_terms: D000818:Animals; D017360:Arabidopsis / Q000378:metabolism; D001665:Binding Sites; D002478:Cells, Cultured; D004331:Drosophila melanogaster / Q000378:metabolism; D004722:Endoribonucleases / Q000378:metabolism; D006801:Humans; D012323:RNA Processing, Post-Transcriptional; D020871:RNA Stability; D012333:RNA, Messenger / Q000378:metabolism*; D012261:Ribonucleoproteins / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000378:metabolism; D020798:Two-Hybrid System Techniques
chemical_list: D012333:RNA, Messenger; D012261:Ribonucleoproteins; D004722:Endoribonucleases