scholarly journals RNA at the surface of phase-separated condensates impacts their size and number

2021 ◽  
Author(s):  
Audrey Cochard ◽  
Marina Garcia-Jove Navarro ◽  
Shunnichi Kashida ◽  
Michel Kress ◽  
Dominique Weil ◽  
...  
Keyword(s):  
Surface Density ◽  
Protein Composition ◽  
Living Cells ◽  
Rna Molecules ◽  
Physical Constraints ◽  
P Bodies ◽  
Intracellular Organization ◽  
Condensate Formation ◽  
Size And Morphology ◽  
Liquid Liquid Phase Separation

Membrane-less organelles, by localizing and regulating complex biochemical reactions, are ubiquitous functional subunits of intracellular organization. They include a variety of nuclear and cytoplasmic ribonucleoprotein (RNP) condensates, such as nucleoli, P-bodies, germ granules and stress granules. While is it now recognized that specific RNA and protein families are critical for the biogenesis of RNP condensates, how these molecular constituents determine condensate size and morphology is unknown. To circumvent the biochemical complexity of endogenous RNP condensates, the use of programmable tools to reconstitute condensate formation with minimal constituents can be instrumental. Here we report a methodology to form RNA-containing condensates in living cells with controlled RNA and protein composition. Our bioengineered condensates are made of ArtiGranule scaffolds undergoing liquid-liquid phase separation in cells and programmed to specifically recruit a unique RNA species. We found that RNAs mainly localized on condensate surface, either as isolated RNA molecules or as a homogenous corona of RNA molecules around the condensate. This simplified system allowed us to demonstrate that the size of the condensates scales with RNA surface density, the higher the RNA density is, the smaller and more frequent the condensates are. Our observations suggest a mechanism based on physical constraints, provided by RNAs localized on condensate surface, that limit condensate growth and coalescence.

Liquid–Liquid Phase Separation in Disease

2019 ◽  
Vol 53 (1) ◽  
pp. 171-194 ◽  
Author(s):  
Simon Alberti ◽  
Dorothee Dormann
Keyword(s):  
Phase Separation ◽  
Living Cells ◽  
Human Diseases ◽  
Therapeutic Interventions ◽  
Rapid Progress ◽  
Disease Mechanisms ◽  
New Concepts ◽  
Condensate Formation ◽  
New Framework ◽  
Liquid Liquid Phase Separation

We have made rapid progress in recent years in identifying the genetic causes of many human diseases. However, despite this recent progress, our mechanistic understanding of these diseases is often incomplete. This is a problem because it limits our ability to develop effective disease treatments. To overcome this limitation, we need new concepts to describe and comprehend the complex mechanisms underlying human diseases. Condensate formation by phase separation emerges as a new principle to explain the organization of living cells. In this review, we present emerging evidence that aberrant forms of condensates are associated with many human diseases, including cancer, neurodegeneration, and infectious diseases. We examine disease mechanisms driven by aberrant condensates, and we point out opportunities for therapeutic interventions. We conclude that phase separation provides a useful new framework to understand and fight some of the most severe human diseases.

scholarly journals Using quantitative reconstitution to investigate multi-component condensates

RNA ◽  
2021 ◽  
pp. rna.079008.121
Author(s):  
Simon L Currie ◽  
Michael K Rosen
Keyword(s):  
Stress Granules ◽  
Lessons Learned ◽  
Sequencing Data ◽  
Processing Bodies ◽  
P Bodies ◽  
Multiple Components ◽  
In Vitro Reconstitution ◽  
Condensate Formation ◽  
Liquid Liquid Phase Separation

Many biomolecular condensates are thought to form via liquid-liquid phase separation (LLPS) of multivalent macromolecules. For those that form through this mechanism, our understanding has benefitted significantly from biochemical reconstitutions of key components and activities. Reconstitutions of RNA-based condensates to date have mostly been based on relatively simple collections of molecules. However, proteomics and sequencing data indicate that natural RNA-based condensates are enriched in hundreds to thousands of different components, and genetic data suggest multiple interactions can contribute to condensate formation to varying degrees. In this perspective we describe recent progress in understanding RNA-based condensates through different levels of biochemical reconstitutions, as a means to bridge the gap between simple in vitro reconstitution and cellular analyses. Complex reconstitutions provide insight into the formation, regulation, and functions of multi-component condensates. We focus on two RNA-protein condensate case studies: stress granules and RNA processing bodies (P bodies), and examine the evidence for cooperative interactions among multiple components promoting LLPS. An important concept emerging from these studies is that composition and stoichiometry regulate biochemical activities within condensates. Based on the lessons learned from stress granules and P bodies we discuss forward-looking approaches to understand the thermodynamic relationships between condensate components, with the goal of developing predictive models of composition and material properties, and their effects on biochemical activities. We anticipate that quantitative reconstitutions will facilitate understanding of the complex thermodynamics and functions of diverse RNA-protein condensates.

scholarly journals Fluctuating Diffusivity of RNA-Protein Particles: Analogy with Thermodynamics

Entropy ◽  
2021 ◽  
Vol 23 (3) ◽  
pp. 333
Author(s):  
Yuichi Itto
Keyword(s):  
Internal Energy ◽  
Messenger Rna ◽  
Living Cells ◽  
Statistical Fluctuation ◽  
Rna Molecules ◽  
Average Value ◽  
Thermodynamic Entropy ◽  
Laws Of Thermodynamics ◽  
Protein Particles ◽  
Quantity Of Heat

A formal analogy of fluctuating diffusivity to thermodynamics is discussed for messenger RNA molecules fluorescently fused to a protein in living cells. Regarding the average value of the fluctuating diffusivity of such RNA-protein particles as the analog of the internal energy, the analogs of the quantity of heat and work are identified. The Clausius-like inequality is shown to hold for the entropy associated with diffusivity fluctuations, which plays a role analogous to the thermodynamic entropy, and the analog of the quantity of heat. The change of the statistical fluctuation distribution is also examined from a geometric perspective. The present discussions may contribute to a deeper understanding of the fluctuating diffusivity in view of the laws of thermodynamics.

In SituHybridization in Living Cells: Detection of RNA Molecules

1997 ◽  
Vol 231 (1) ◽  
pp. 226-233 ◽  
Author(s):  
S. Paillasson ◽  
M. Van De Corput ◽  
R.W. Dirks ◽  
H.J. Tanke ◽  
M. Robert-Nicoud ◽  
...  
Keyword(s):  
Living Cells ◽  
Rna Molecules ◽  
In Situhybridization

scholarly journals Effects on protein synthesis of injecting synthetic polyribonucleotides into living cells

1974 ◽  
Vol 144 (1) ◽  
pp. 11-19 ◽  
Author(s):  
Hugh Woodland ◽  
Sarah E. Ayers
Keyword(s):  
Protein Synthesis ◽  
Xenopus Laevis ◽  
Potent Inhibitor ◽  
Living Cells ◽  
Initiation Factor ◽  
Rna Molecules ◽  
Endogenous Protein ◽  
Limited Supply ◽  
Inhibitor Of Protein Synthesis ◽  
Haemoglobin Synthesis

Micro-injection into the oocytes and eggs of Xenopus laevis was used to ascertain the effects of synthetic polyribonucleotides on protein synthesis in living cells. Poly(U) and poly(A) were not translated detectably, nor did they change the rate of endogenous protein synthesis. The same was true of poly(G,U), poly(A,G,U), poly(A,C,G,U), G-U-G-(U)n, A-(U)n and AUG. In contrast, A-U-G-(U)n was a potent inhibitor of protein synthesis in the cell. This might be because it is initiated normally but lacks a termination codon, or because it inhibits the translation of other molecules in some way not dependent on its normal initiation. Poly(G,U), poly(A,G,U) and poly(A,C,G,U) inhibited haemoglobin synthesis when they were injected into the oocyte with haemoglobin mRNA. The synthetic polyribonucleotides did not inhibit the translation of the natural mRNA when the two sorts of molecules were injected at different times. It is suggested that the synthetic RNA molecules compete with the natural mRNA for a pre-initiation factor in limited supply.

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.

Physical Principles Underlying the Complex Biology of Intracellular Phase Transitions

2020 ◽  
Vol 49 (1) ◽  
pp. 107-133 ◽  
Author(s):  
Jeong-Mo Choi ◽  
Alex S. Holehouse ◽  
Rohit V. Pappu
Keyword(s):  
Phase Transitions ◽  
Rheological Properties ◽  
Recent Work ◽  
Driving Forces ◽  
Rna Molecules ◽  
Associative Polymers ◽  
Different Types ◽  
Condensate Formation ◽  
The Impact ◽  
Physical Principles

Many biomolecular condensates appear to form via spontaneous or driven processes that have the hallmarks of intracellular phase transitions. This suggests that a common underlying physical framework might govern the formation of functionally and compositionally unrelated biomolecular condensates. In this review, we summarize recent work that leverages a stickers-and-spacers framework adapted from the field of associative polymers for understanding how multivalent protein and RNA molecules drive phase transitions that give rise to biomolecular condensates. We discuss how the valence of stickers impacts the driving forces for condensate formation and elaborate on how stickers can be distinguished from spacers in different contexts. We touch on the impact of sticker- and spacer-mediated interactions on the rheological properties of condensates and show how the model can be mapped to known drivers of different types of biomolecular condensates.

scholarly journals Intrinsic Disorder-Based Emergence in Cellular Biology: Physiological and Pathological Liquid-Liquid Phase Transitions in Cells

Polymers ◽  
2019 ◽  
Vol 11 (6) ◽  
pp. 990 ◽  
Author(s):  
April L. Darling ◽  
Boris Y. Zaslavsky ◽  
Vladimir N. Uversky
Keyword(s):  
Phase Transitions ◽  
Phase Separation ◽  
Liquid Phase ◽  
Intrinsic Disorder ◽  
Living Cells ◽  
Cellular Biology ◽  
Physiological Functions ◽  
Pathological Conditions ◽  
Liquid Liquid Phase Separation ◽  
In Cells

The visible outcome of liquid-liquid phase transitions (LLPTs) in cells is the formation and disintegration of various proteinaceous membrane-less organelles (PMLOs). Although LLPTs and related PMLOs have been observed in living cells for over 200 years, the physiological functions of these transitions (also known as liquid-liquid phase separation, LLPS) are just starting to be understood. While unveiling the functionality of these transitions is important, they have come into light more recently due to the association of abnormal LLPTs with various pathological conditions. In fact, several maladies, such as various cancers, different neurodegenerative diseases, and cardiovascular diseases, are known to be associated with either aberrant LLPTs or some pathological transformations within the resultant PMLOs. Here, we will highlight both the physiological functions of cellular liquid-liquid phase transitions as well as the pathological consequences produced through both dysregulated biogenesis of PMLOs and the loss of their dynamics. We will also discuss the potential downstream toxic effects of proteins that are involved in pathological formations.

scholarly journals Analysis of membrane proteins localizing to the inner nuclear envelope in living cells

2016 ◽  
Vol 215 (4) ◽  
pp. 575-590 ◽  
Author(s):  
Christine J. Smoyer ◽  
Santharam S. Katta ◽  
Jennifer M. Gardner ◽  
Lynn Stoltz ◽  
Scott McCroskey ◽  
...  
Keyword(s):  
Membrane Proteins ◽  
Nuclear Membrane ◽  
Fluorescent Protein ◽  
Protein Localization ◽  
Transmembrane Protein ◽  
Protein Composition ◽  
Living Cells ◽  
Correlation Spectroscopy ◽  
Green Fluorescent ◽  
And Function

Understanding the protein composition of the inner nuclear membrane (INM) is fundamental to elucidating its role in normal nuclear function and in disease; however, few tools exist to examine the INM in living cells, and the INM-specific proteome remains poorly characterized. Here, we adapted split green fluorescent protein (split-GFP) to systematically localize known and predicted integral membrane proteins in Saccharomyces cerevisiae to the INM as opposed to the outer nuclear membrane. Our data suggest that components of the endoplasmic reticulum (ER) as well as other organelles are able to access the INM, particularly if they contain a small extraluminal domain. By pairing split-GFP with fluorescence correlation spectroscopy, we compared the composition of complexes at the INM and ER, finding that at least one is unique: Sbh2, but not Sbh1, has access to the INM. Collectively, our work provides a comprehensive analysis of transmembrane protein localization to the INM and paves the way for further research into INM composition and function.

scholarly journals RNAs as Regulators of Cellular Matchmaking

2021 ◽  
Vol 8 ◽  
Author(s):  
Nikita Fernandes ◽  
J. Ross Buchan
Keyword(s):  
Protein Localization ◽  
Mrna Translation ◽  
Rna Molecules ◽  
P Bodies ◽  
Coding Regions ◽  
Cytoplasmic Rna ◽  
Chromosome Topology ◽  
Future Challenges ◽  
Non Coding Rnas ◽  
Nuclear Processes

RNA molecules are increasingly being identified as facilitating or impeding the interaction of proteins and nucleic acids, serving as so-called scaffolds or decoys. Long non-coding RNAs have been commonly implicated in such roles, particularly in the regulation of nuclear processes including chromosome topology, regulation of chromatin state and gene transcription, and assembly of nuclear biomolecular condensates such as paraspeckles. Recently, an increased awareness of cytoplasmic RNA scaffolds and decoys has begun to emerge, including the identification of non-coding regions of mRNAs that can also function in a scaffold-like manner to regulate interactions of nascently translated proteins. Collectively, cytoplasmic RNA scaffolds and decoys are now implicated in processes such as mRNA translation, decay, protein localization, protein degradation and assembly of cytoplasmic biomolecular condensates such as P-bodies. Here, we review examples of RNA scaffolds and decoys in both the nucleus and cytoplasm, illustrating common themes, the suitability of RNA to such roles, and future challenges in identifying and better understanding RNA scaffolding and decoy functions.

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