Cells organize biochemical processes into biological condensates. P-bodies are cytoplasmic condensates that are enriched in enzymes important for mRNA degradation and have been identified as sites of both storage and decay. How these opposing outcomes can be achieved in condensates remains unresolved. mRNA decapping immediately precedes degradation, and the Dcp1/Dcp2 decapping complex is enriched in P-bodies. Here, we show that Dcp1/Dcp2 activity is modulated in condensates and depends on the interactions promoting phase separation. We find that Dcp1/Dcp2 phase separation stabilizes an inactive conformation in Dcp2 to inhibit decapping. The activator Edc3 causes a conformational change in Dcp2 and rewires the protein–protein interactions to stimulate decapping in condensates. Disruption of the inactive conformation dysregulates decapping in condensates. Our results indicate that the regulation of enzymatic activity in condensates relies on a coupling across length scales ranging from microns to ångstroms. We propose that this regulatory mechanism may control the functional state of P-bodies and related phase-separated compartments.
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Activation of L-lactate oxidase by the formation of enzyme assemblies through liquid–liquid phase separation
Scientific Reports Open Access 25 January 2023
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We thank the Center for Advanced Light Microscopy at UCSF for guidance and technical assistance in collecting microscopy data. We also thank M. Warminski and A. Mlynarska-Cieslak (University of Warsaw) for providing reagents for RNA labeling and M. Kelly for assistance through the UCSF NMR Facility. We thank C. Freund, A. Manglik, D. Brown and members of the J.D.G. lab for experimental guidance and many helpful discussions. We extend thanks to G. Narlikar, S. Floor and S. Sanulli for useful discussions in writing the manuscript. Use of the spinning disk confocal microscope was supported by the US National Institutes of Health (1S10OD017993-01A1). This work was supported by the US National Institutes of Health (R01 GM078360 to J.D.G.), the Foundation for Polish Science (TEAM/2016-2/13 to J.J.), the National Science Centre, Poland (UMO-2018/31/B/ST5/03821 to J.K.), a Moritz-Heyman UCSF Discovery Fellowship (to R.W.T.) and an ARCS Foundation Fellowship (to R.W.T.).
The authors declare no competing interests.
Peer review information Nature Chemical Biology thanks Ross Buchan and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.
Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
a, Overview of the labelling procedure, IVT – in vitro transcription, c.t. – co-transcriptional, p.t. – post-transcriptional; b, structures of the reagents used for the 5’ and 3’ end labelling; c, Analysis of purified 5’ capped & labelled RNA after IVT: lane 1 – reference uncapped RNA, lane 2 – RNA capped co-transcriptionally with fluorescent cap analog (FAM-m7Gp3AmpG). d, Labelling of the 3’ end of FAM-m7Gp3AmpG-RNA with pAp-SCy5 to yield dually labelled probe: lane 3 – crude dually labelled RNA after purification; lane 4 – HPLC-purified RNA probe. e, f, Co-localization of m7G cap (fluorescein) and RNA body (Cy5) in Dcp1/Dcp2ext/Edc3 condensates over twenty minutes demonstrates decapping does not occur in the absence of Mg2+, which is required for catalysis. g, Excess RNA slows initial rate of decapping two-fold in droplets formed with 1 μM Dcp1/Dcp2ext and 15 μM Edc3. Total RNA concentration is 100 nM when limiting and 20 μM when in excess. Representative micrographs and data in f, g are presented as mean ± s.e.m. for twenty droplets examined in two independent experiments with similar results. Error bars are not depicted when smaller than the data points.
Extended Data Fig. 2 Edc3 sequesters Dcp1/Dcp2ext in condensates to cooperatively activate decapping.
a, Enhancement of decapping by Edc3 Lsm domain occurs in the absence of condensates. C-terminal Edc3 YjeF N domain does not stimulate Dcp1/Dcp2ext activity or cause phase separation. Activation by full-length Edc3 is reproduced from Fig. 4c for comparison. Two independent experiments are shown. Representative micrographs are from three independent experiments with similar results. b, Cooperativity of activation by dimeric Edc3 is five-fold greater than the Lsm domain. Hill coefficients are reported as mean ± standard error from fitting two independent experiments shown in a. c, Depletion of Dcp1/Dcp2ext from the dilute phase at increasing concentrations of Edc3 visualized by SDS-PAGE. (Top) Instant Blue staining reveals total protein remaining in dilute phase following pelleting of liquid droplets. (Bottom) In-gel fluorescence of Cy5-labelled Dcp2 shows its Edc3-dependent disappearance from the solution. d, Quantification of Dcp1/Dcp2ext from analysis of Dcp2 stained with Instant Blue or measured by in-gel Cy5 emission. Cy5 fluorescence is reproduced from Fig. 3d for comparison. Representative SDS-PAGE and quantification are from two independent experiments with data presented as mean ± s.e.m. Error bars are not shown when smaller than the data points.
a, Superstoichiometric Edc3 enriches Dcp1/Dcp2ext in droplets and causes causes droplets to be more sensitive to presence of RNA. Images below graph correspond to representative Cy5-labelled Dcp1/Dcp2ext micrographs and data presented are mean ± s.e.m. for five independent experiments with similar results. b, Enrichment of FAM-29mer RNA in droplets of varying Dcp1/Dcp2ext and Edc3 concentrations. Representative micrographs and data presented are mean ± s.e.m. for ten independent experiments with similar results. c, Edc3 increases the mobile fraction of RNA in droplets. Dcp1/Dcp2ext concentration is 40 μM in absence and 5 μM in presence of 80 μM Edc3. Data presented are mean ± s.e.m. for twenty recovery profiles collected over two independent experiments. Error bars are not depicted when smaller than the data point.
Extended Data Fig. 4 Several resonances in the catalytic domain of Dcp2 report on the inactive—precatalytic equilibrium.
a, 1H/13C-methyl resonances in Dcp2 constructs predominantly fall along a linear trajectory (dotted line), indicative of fast interconversion between the inactive and precatalytic states on the NMR timescale. Resonances for Ala 227 Cβ, Ile 223δ1, and Ile 196δ1 were used in addition to Ile 102δ1 (Fig. 5b) to calculate the relative population of the inactive state due to the observance of resonances for all constructs strictly lying along a linear trajectory.
Extended Data Fig. 5 Dcp1/Dcp2ext conformational equilibria is important for substrate recognition, liquid-like behavior, and proper regulation of decapping in condensates.
a, FP curves for various Dcp2 constructs binding to U30mer RNA. Data are normalized to the span between minimum and maximum mP values for each protein tested. Dcp1/Dcp2ext was normalized to span between its minimum and average maximum for all proteins tested. Data are presented as mean ± s.e.m. for three independent experiments and error bars are not show when smaller than the data point. b, Fusion of Dcp1/Dcp2ext(Y220G) condensates occurs slower than wild-type droplets of similar size. Time (τ) data presented are from fits of exponential decrease in droplet length following initial fusion event and error represents standard error of the fit and are not depicted when smaller than the data point. Representative micrographs are from three independent experiments with similar results. c, Decapping of dual-labeled RNA substrate by Dcp1/Dcp2ext(Y220G) by fluorescence microscopy. Representative micrographs are from twenty droplets collected over two independent experiments with similar results. d, The Y220G mutation does not affect the cooperativity of activation by Edc3 but increases the K1/2 of activation three-fold. Hill coefficients and K1/2 are presented as mean ± s.e.m. for experimental fits from two independent experiments shown in Fig. 5g.
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Tibble, R.W., Depaix, A., Kowalska, J. et al. Biomolecular condensates amplify mRNA decapping by biasing enzyme conformation. Nat Chem Biol 17, 615–623 (2021). https://doi.org/10.1038/s41589-021-00774-x
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