title: In vitro reconstitution of a cellular phase-transition process that involves the mRNA decapping machinery.
abstract: In eukaryotic cells, components of the 5' to 3' mRNA degradation machinery can undergo a rapid phase transition. The resulting cytoplasmic foci are referred to as processing bodies (P-bodies). The molecular details of the self-aggregation process are, however, largely undetermined. Herein, we use a bottom-up approach that combines NMR spectroscopy, isothermal titration calorimetry, X-ray crystallography, and fluorescence microscopy to probe if mRNA degradation factors can undergo phase transitions in vitro. We show that the Schizosaccharomyces pombe Dcp2 mRNA decapping enzyme, its prime activator Dcp1, and the scaffolding proteins Edc3 and Pdc1 are sufficient to reconstitute a phase-separation process. Intermolecular interactions between the Edc3 LSm domain and at least 10 helical leucine-rich motifs in Dcp2 and Pdc1 build the core of the interaction network. We show that blocking of these interactions interferes with the clustering behavior, both in vitro and in vivo.
authors.affiliation: Max Planck Institute for Developmental Biology, Spemannstrasse 35, 72076 Tübingen (Germany).
authors.forename: Simon A
authors.initials: SA
authors.lastname: Fromm
authors.forename: Julia
authors.initials: J
authors.lastname: Kamenz
authors.forename: Erik R
authors.initials: ER
authors.lastname: Nöldeke
authors.forename: Ancilla
authors.initials: A
authors.lastname: Neu
authors.forename: Georg
authors.initials: G
authors.lastname: Zocher
authors.forename: Remco
authors.initials: R
authors.lastname: Sprangers
mesh_terms: D018360:Crystallography, X-Ray; D004722:Endoribonucleases / Q000378:metabolism*; D066298:In Vitro Techniques; D009682:Magnetic Resonance Spectroscopy; D008958:Models, Molecular; D011485:Protein Binding; D012333:RNA, Messenger / Q000378:metabolism*; D012568:Schizosaccharomyces / Q000201:enzymology*; D029702:Schizosaccharomyces pombe Proteins / Q000235:genetics / Q000378:metabolism
chemical_list: C508496:Dcp2 protein, S pombe; D012333:RNA, Messenger; D029702:Schizosaccharomyces pombe Proteins; C030052:mRNA decapping enzymes; D004722:Endoribonucleases
keywords: NMR spectroscopy; phase transitions; processing bodies; protein-protein interactions; self-assembly