scholarly journals The arrested state of processing bodies supports mRNA regulation in early development

2021 ◽  
Author(s):  
M. Sankaranarayanan ◽  
Ryan J. Emenecker ◽  
Marcus Jahnel ◽  
Irmela R. E. A. Trussina ◽  
Matt Wayland ◽  
...  
Keyword(s):  
Physical Properties ◽  
Electrostatic Interactions ◽  
Processing Bodies ◽  
P Bodies ◽  
Intrinsically Disordered ◽  
Intrinsically Disordered Regions ◽  
Dead Box ◽  
Premature Release ◽  
Liquid Liquid Phase Separation

ABSTRACTBiomolecular condensates that form via liquid-liquid phase separation can exhibit diverse physical states. Despite considerable progress, the relevance of condensate physical states forin vivobiological function remains limited. Here, we investigated the physical properties ofin vivoprocessing bodies (P bodies) and their impact on mRNA storage in matureDrosophilaoocytes. We show that the conserved DEAD-box RNA helicase Me31B forms P body condensates which adopt a less dynamic, arrested physical state. We demonstrate that structurally distinct proteins and hydrophobic and electrostatic interactions, together with RNA and intrinsically disordered regions, regulate the physical properties of P bodies. Finally, using live imaging, we show that the arrested state of P bodies is required to prevent the premature release ofbicoid(bcd) mRNA, a body axis determinant, and that P body dissolution leads tobcdrelease. Together, this work establishes a role for arrested states of biomolecular condensates in regulating cellular function in a developing organism.

scholarly journals Pat1 promotes processing body assembly by enhancing the phase separation of the DEAD-box ATPase Dhh1 and RNA

2018 ◽  
Author(s):  
Ruchika Sachdev ◽  
Maria Hondele ◽  
Miriam Linsenmeier ◽  
Pascal Vallotton ◽  
Christopher F. Mugler ◽  
...  
Keyword(s):  
Phase Separation ◽  
Direct Interaction ◽  
Liquid Droplets ◽  
Processing Bodies ◽  
Dead Box ◽  
The Dead ◽  
Processing Body ◽  
Liquid Liquid Phase Separation

AbstractProcessing bodies (PBs) are cytoplasmic mRNP granules that assemble via liquid-liquid phase separation and are implicated in the decay or storage of mRNAs. How PB assembly is regulated in cells remains unclear. We recently identified the ATPase activity of the DEAD-box protein Dhh1 as a key regulator of PB dynamics and demonstrated that Not1, an activator of the Dhh1 ATPase and member of the CCR4-NOT deadenylase complex inhibits PB assembly in vivo [Mugler et al., 2016]. Here, we show that the PB component Pat1 antagonizes Not1 and promotes PB assembly via its direct interaction with Dhh1. Intriguingly, in vivo PB dynamics can be recapitulated in vitro, since Pat1 enhances the phase separation of Dhh1 and RNA into liquid droplets, whereas Not1 reverses Pat1-Dhh1-RNA condensation. Overall, our results uncover a function of Pat1 in promoting the multimerization of Dhh1 on mRNA, thereby aiding the assembly of large multivalent mRNP granules that are PBs.

scholarly journals Pat1 promotes processing body assembly by enhancing the phase separation of the DEAD-box ATPase Dhh1 and RNA

eLife ◽  
2019 ◽  
Vol 8 ◽  
Author(s):  
Ruchika Sachdev ◽  
Maria Hondele ◽  
Miriam Linsenmeier ◽  
Pascal Vallotton ◽  
Christopher F Mugler ◽  
...  
Keyword(s):  
Phase Separation ◽  
Direct Interaction ◽  
Liquid Droplets ◽  
Processing Bodies ◽  
Dead Box ◽  
The Dead ◽  
Processing Body ◽  
Liquid Liquid Phase Separation

Processing bodies (PBs) are cytoplasmic mRNP granules that assemble via liquid–liquid phase separation and are implicated in the decay or storage of mRNAs. How PB assembly is regulated in cells remains unclear. Previously, we identified the ATPase activity of the DEAD-box protein Dhh1 as a key regulator of PB dynamics and demonstrated that Not1, an activator of the Dhh1 ATPase and member of the CCR4-NOT deadenylase complex inhibits PB assembly in vivo (Mugler et al., 2016). Here, we show that the PB component Pat1 antagonizes Not1 and promotes PB assembly via its direct interaction with Dhh1. Intriguingly, in vivo PB dynamics can be recapitulated in vitro, since Pat1 enhances the phase separation of Dhh1 and RNA into liquid droplets, whereas Not1 reverses Pat1-Dhh1-RNA condensation. Overall, our results uncover a function of Pat1 in promoting the multimerization of Dhh1 on mRNA, thereby aiding the assembly of large multivalent mRNP granules that are PBs.

scholarly journals Thermodynamic and sequential characteristics of phase separation and droplet formation for an intrinsically disordered region/protein ensemble

2021 ◽  
Vol 17 (3) ◽  
pp. e1008672
Author(s):  
Wen-Ting Chu ◽  
Jin Wang
Keyword(s):  
Phase Separation ◽  
High Temperatures ◽  
Electrostatic Interactions ◽  
Sequence Length ◽  
Intrinsically Disordered ◽  
Dynamics Simulations ◽  
Conventional Behavior ◽  
Liquid Liquid Phase Separation

Liquid–liquid phase separation (LLPS) of some IDPs/IDRs can lead to the formation of the membraneless organelles in vitro and in vivo, which are essential for many biological processes in the cell. Here we select three different IDR segments of chaperon Swc5 and develop a polymeric slab model at the residue-level. By performing the molecular dynamics simulations, LLPS can be observed at low temperatures even without charge interactions and disappear at high temperatures. Both the sequence length and the charge pattern of the Swc5 segments can influence the critical temperature of LLPS. The results suggest that the effects of the electrostatic interactions on the LLPS behaviors can change significantly with the ratios and distributions of the charged residues, especially the sequence charge decoration (SCD) values. In addition, three different forms of swc conformation can be distinguished on the phase diagram, which is different from the conventional behavior of the free IDP/IDR. Both the packed form (the condensed-phase) and the dispersed form (the dilute-phase) of swc chains are found to be coexisted when LLPS occurs. They change to the fully-spread form at high temperatures. These findings will be helpful for the investigation of the IDP/IDR ensemble behaviors as well as the fundamental mechanism of the LLPS process in bio-systems.

scholarly journals Cavin1 intrinsically disordered domains are essential for fuzzy electrostatic interactions and caveola formation

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Vikas A. Tillu ◽  
James Rae ◽  
Ya Gao ◽  
Nicholas Ariotti ◽  
Matthias Floetenmeyer ◽  
...  
Keyword(s):  
Electrostatic Interactions ◽  
Membrane Lipid ◽  
Membrane Curvature ◽  
Caveolin 1 ◽  
Intrinsically Disordered ◽  
Intrinsically Disordered Regions ◽  
Solution Generation ◽  
Endosomal System ◽  
Disordered Regions

AbstractCaveolae are spherically shaped nanodomains of the plasma membrane, generated by cooperative assembly of caveolin and cavin proteins. Cavins are cytosolic peripheral membrane proteins with negatively charged intrinsically disordered regions that flank positively charged α-helical regions. Here, we show that the three disordered domains of Cavin1 are essential for caveola formation and dynamic trafficking of caveolae. Electrostatic interactions between disordered regions and α-helical regions promote liquid-liquid phase separation behaviour of Cavin1 in vitro, assembly of Cavin1 oligomers in solution, generation of membrane curvature, association with caveolin-1, and Cavin1 recruitment to caveolae in cells. Removal of the first disordered region causes irreversible gel formation in vitro and results in aberrant caveola trafficking through the endosomal system. We propose a model for caveola assembly whereby fuzzy electrostatic interactions between Cavin1 and caveolin-1 proteins, combined with membrane lipid interactions, are required to generate membrane curvature and a metastable caveola coat.

scholarly journals G-quadruplex DNA inhibits unwinding activity but promotes liquid–liquid phase separation by the DEAD-box helicase Ded1p

2021 ◽  
Author(s):  
Jun Gao ◽  
Zhaofeng Gao ◽  
Andrea A. Putnam ◽  
Alicia K. Byrd ◽  
Sarah L. Venus ◽  
...  
Keyword(s):  
Phase Separation ◽  
Liquid Phase ◽  
Liquid Phase Separation ◽  
Intrinsically Disordered ◽  
Dead Box ◽  
G4 Dna ◽  
G Quadruplex ◽  
The Dead ◽  
Liquid Liquid Phase Separation

G-quadruplex (G4) DNA inhibits RNA unwinding activity but promotes liquid–liquid phase separation of the DEAD-box helicase Ded1p in vitro and in cells. This highlights multifaceted effects of G4DNA on an enzyme with intrinsically disordered domains.

scholarly journals Basic Residue Clusters in Intrinsically Disordered Regions of Peripheral Membrane Proteins: Modulating 2D Diffusion on Cell Membranes

Physchem ◽  
2021 ◽  
Vol 1 (2) ◽  
pp. 152-162
Author(s):  
Miquel Pons
Keyword(s):  
Membrane Proteins ◽  
Electrostatic Interactions ◽  
Lipid Membrane ◽  
Conformational Ensemble ◽  
Basic Residue ◽  
Intrinsically Disordered ◽  
Intrinsically Disordered Regions ◽  
Peripheral Membrane Proteins ◽  
Charged Residues ◽  
Disordered Regions

A large number of peripheral membrane proteins transiently interact with lipids through a combination of weak interactions. Among them, electrostatic interactions of clusters of positively charged amino acid residues with negatively charged lipids play an important role. Clusters of charged residues are often found in intrinsically disordered protein regions, which are highly abundant in the vicinity of the membrane forming what has been called the disordered boundary of the cell. Beyond contributing to the stability of the lipid-bound state, the pattern of charged residues may encode specific interactions or properties that form the basis of cell signaling. The element of this code may include, among others, the recognition, clustering, and selective release of phosphatidyl inositides, lipid-mediated protein-protein interactions changing the residence time of the peripheral membrane proteins or driving their approximation to integral membrane proteins. Boundary effects include reduction of dimensionality, protein reorientation, biassing of the conformational ensemble of disordered regions or enhanced 2D diffusion in the peri-membrane region enabled by the fuzzy character of the electrostatic interactions with an extended lipid membrane.

scholarly journals Tuning levels of low-complexity domain interactions to modulate endogenous oncogenic transcription

2021 ◽  
Author(s):  
Shasha Chong ◽  
Thomas G. W. Graham ◽  
Claire Dugast-Darzacq ◽  
Gina M. Dailey ◽  
Xavier Darzacq ◽  
...  
Keyword(s):  
Single Molecule ◽  
Target Genes ◽  
Gene Activation ◽  
Low Complexity ◽  
Intrinsically Disordered ◽  
Human Genes ◽  
Domain Interactions ◽  
Bona Fide ◽  
Liquid Liquid Phase Separation

Gene activation by mammalian transcription factors (TFs) requires dynamic, multivalent, and selective interactions of their intrinsically disordered low-complexity domains (LCDs), but how such interactions mediate transcription remains unclear. It has been proposed that extensive LCD-LCD interactions culminating in liquid-liquid phase separation (LLPS) of TFs is the dominant mechanism underlying transactivation. Here, we investigated how tuning the amount and localization of LCD-LCD interactions in vivo affects transcription of endogenous human genes. Quantitative single-cell and single-molecule imaging reveals that the oncogenic TF EWS/FLI1 requires a finely tuned range of LCD-LCD interactions to efficiently activate target genes. Modest or more dramatic increases in LCD-LCD interactions toward putative LLPS repress EWS/FLI1-driven transcription in patient cells. Likewise, ectopically creating LCD-LCD interactions to sequester EWS/FLI1 into a bona fide LLPS compartment, the nucleolus, inhibits EWS/FLI1-driven transcription and oncogenic transformation. Our findings reveal fundamental principles underlying LCD-mediated transcription and suggest mislocalizing specific LCD-LCD interactions as a novel therapeutic strategy for targeting disease-causing TFs.

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