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.

Keywords: NMR spectroscopy; phase transitions; processing bodies; protein-protein interactions; self-assembly.

Figure 1

The seven HLMs in Dcp2 bind to the Edc3 LSm domain with a wide range of affinities. A) Schematic representation of S. pombe Dcp1 (EVH1 domain, yellow), Dcp2 (regulatory light green, catalytic dark green, HLMs red, proline-rich mid domain white, C-terminus gray) and Edc3 (LSm dark blue, FDF motif orange, YjeF N light blue). All used protein constructs are listed in Supporting Information Table S1 A. B) ¹H–¹⁵N correlation spectra of the binding between the monomeric Edc3 LSm domain and the seven isolated Dcp2 HLMs. Black: free ¹⁵N labeled Edc3 LSm domain, Red: the Edc3 LSm domain in the presence of a fivefold excess of the individual Dcp2 HLMs. The boxed region in the top left panel is shown in all other spectra. Residues R38 and L58 are highlighted with ovals to indicate that the HLMs induce chemical shift perturbations to a different extent. C) ITC graphs for the binding of the Edc3 LSm domain to the Dcp2 HLM-1, HLM-C1 and HLM-C2 sequences. The best fit is drawn with a red line and the extracted K_d values including the error (standard deviation) are indicated. Deviations from n=1.0, where n refers to the stoichiometry, result from small inaccuracies in the protein-concentration determination. D) Overview of the determined K_d values (error in parenthesis) for the Dcp2 HLM:Edc3 interactions (n.d.: not determinable with ITC).

Figure 2

In vitro phase transitions of purified Edc3 and Dcp2. A) Phase transition of 50 μm Dcp2 553-741 and 150 μm Edc3 (doped 1:100 with Edc3-OregonGreen). Left: bright field (BF) channel; middle: Oregon Green (OG) channel; right: merge. B) Phase diagrams of phase transition of Edc3 together with Dcp2 553–741 (that contains 4 HLM sequences; left) or Dcp2 242–741ΔMID (that contains 5 HLM sequences). Given concentrations are modular concentrations, for example, 50 μm of Dcp2 553–741 is 200 μm HLMs, because 4 HLMs are in the Dcp2 553–741 construct. Modular LSm concentration is identical with total Edc3 concentration as an Edc3 monomer has one LSm domain. Occurrence of phase transition at a given condition is color coded; blue=no phase transition; light blue=beginning phase transition; green=clear phase transition. Green lines indicate progression of the phase boundaries. The red encircled condition is shown in (A) and (C). C) The in vitro droplets are highly dynamic and fuse over time. The time-scale is indicated below the OG channel pictures. D) Droplets formed by 25 μm Dcp1:Dcp2ΔMID and 100 μm Edc3 (doped 1:100 with Edc3-OG). Left: BF channel, Right: OG channel. E) Droplets formed by 25 μm Dcp2 242–741ΔMID and 100 μm Edc3 disappear by addition of increasing amounts of Dcp2 242–291 (containing HLM-1) (5 μm, 10 μm, 25 μm, 50 μm). Scale bars: 50 μm.

Figure 3

Effect of HLM-1 expression on Edc3 localization in vivo. A,B) Fluorescent micrographs of S. pombe expressing mCherry and GFP tagged versions of Edc3 and Dcp2 (A) or Lsm7 (B), respectively. Without HLM-1 overexpression Edc3 and Dcp2 are enriched into P-bodies (top row). Upon overexpression (adh1 promoter) of HLM-1 Edc3 no longer localizes to P-bodies but is diffusely spread in the cell (bottom row). Dcp2-GFP (A) and Lsm7 (B) still localize to P-bodies upon overexpression of HLM-1, indicating that interference with the Edc3:HLM interactions does not generally disturb P-body formation. Scale bar: 5 μm.

Figure 4

Pdc1 contains three HLMs at the N-terminus that bind to Edc3 with different affinities. A) Domain organization of the Pdc1 protein (N-terminal HLMs cyan, WD40 repeats purple, coiled-coil orange, Ge-1_C-like domain (see below) pink). B) ¹H–¹⁵N correlation spectra of the monomeric ¹⁵N labeled Edc3 LSm domain in the absence (black) and presence (cyan) of the individual Pdc1 HLMs. The boxed region in the top left panel of Figure 1 B is shown, ovals highlight two specific residues (see above). C) ITC graph of the Edc3 LSm domain binding to HLM-N2 from Pdc1. The best fit is drawn with a cyan line and the extracted K_d value is indicated. D) Overview of all K_d values (error in parentheses) determined for binding of Pdc1 HLMs to Edc3 LSm (n.d., not determinable with ITC). E) In vitro phase separation of designed dimeric Pdc1 construct that mimics full length Pdc1 (50 μm) together with Edc3 (150 μm; doped 1:100 with Edc3-OregonGreen). Scale bar, 50 μm.

Figure 5

A) Ribbon diagram of the crystal structure of the Pdc1 Ge-1_C domain. Residues that interact with the decapping complex are highlighted in olive. B) NMR spectra of the Pdc1 Ge-1_C domain in the absence (black) and presence (olive) of the Dcp1:Dcp2 decapping complex that contains only the Dcp2 regulatory region. Resonances that experience chemical shift changes are labeled with the residue number. “*” refers to a resonance that is not assigned. C) The building blocks of the network of intermolecular interactions that leads to phase separation: One Edc3 dimer can interact with two decapping enzymes (left top), with two Pdc1 proteins (right top) or with one decapping complex and one Pdc1 dimer (bottom left). In addition one Pdc1 dimer can interact with two decapping complexes (bottom right). D) Schematic representation of how the building blocks displayed in (C) can be extended into an indefinite network of interactions. Edc3 blue, Dcp1 yellow, Dcp2 green, Pdc1 pink. These intermolecular interactions can result in phase separation (see Figures 2 and 4).