InVitro 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 invitro. 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 Edc3LSm domain and at least 10helical 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 invitro and invivo. © 2014 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.