Quantitative reconstitution of yeast RNA processing bodies

Abstract

Many biomolecular condensates appear to form through liquid–liquid phase separation (LLPS). Individual condensate components can often undergo LLPS in vitro, capturing some features of the native structures. However, natural condensates contain dozens of components with different concentrations, dynamics, and contributions to compartment formation. Most biochemical reconstitutions of condensates have not benefited from quantitative knowledge of these cellular features nor attempted to capture natural complexity. Here, we build on prior quantitative cellular studies to reconstitute yeast RNA processing bodies (P bodies) from purified components. Individually, five of the seven highly concentrated P-body proteins form homotypic condensates at cellular protein and salt concentrations, using both structured domains and intrinsically disordered regions. Combining the seven proteins together at their cellular concentrations with RNA yields phase-separated droplets with partition coefficients and dynamics of most proteins in reasonable agreement with cellular values. RNA delays the maturation of proteins within and promotes the reversibility of, P bodies. Our ability to quantitatively recapitulate the composition and dynamics of a condensate from its most concentrated components suggests that simple interactions between these components carry much of the information that defines the physical properties of the cellular structure.

Advanced Surface Passivation for High-Sensitivity Studies of Biomolecular Condensates

“…Many condensates form through liquid-liquid phase separation (LLPS) of multivalent macromolecules (1, 3). Significant advancements in our understanding of condensates have derived from biochemical reconstitution with fluorescence microscopy detection (4-7). Undesirable interactions between condensates and glass surfaces represent a significant methodological challenge in these experiments.…”

Amyloid Beta Oligomers Accelerate ATP-Dependent Phase Separation of miRNA-Bound Ago2 to RNA Processing Bodiesin vitro

“…A recent study showed a quantitative in vitro reconstitution of yeast RNA processing bodies (P-bodies) by combining seven proteins, which are found at high concentrations in PBs, and also RNA at concentrations which are normally seen at the cellular level and it results in the formation of phase separated droplets, quantitatively resembling the protein partition coefficient and dynamics of cellular measurements. Here, only physiological/cellular conditions are used for this in vitro reconstitution and the role of RNA in P-body assembly was not completely understood (42). In our study we tried to establish such an in vitro system which can be manipulated in many ways mimicking cellular conditions to reveal the role of different internal or externally added factors on the in vitro P-body assembly process.…”

A stable microtubule bundle formed through an orchestrated multistep process controls quiescence exit

“…Among these modifications, cellular volume reduction increases molecular crowding ( Joyner et al, 2016 ) and pH acidification both increases viscosity and causes changes in macromolecule surface charges ( Charruyer and Ghadially, 2018 ; Jacquel et al, 2021 ; Munder et al, 2016 ; Persson et al, 2020 ). Much evidence suggests that these modifications act as triggers for the auto-assembly of several types of enzyme-containing granules ( Munder et al, 2016 ; Petrovska et al, 2014 ; Rabouille and Alberti, 2017 ) or complex structures such as P-bodies and Proteasome Storage Granules ( Peters et al, 2013 ; Jacquel et al, 2021 ; Currie et al, 2023 ). Recently, Molines and colleagues demonstrated that cytoplasmic viscosity modulates MT dynamics in vivo ( Molines et al, 2022 ).…”