Coming Together: RNAs and Proteins Assemble under the Single-Molecule Fluorescence Microscope

Biological liquid-liquid phase separation has gained considerable attention in recent years as a driving force for the assembly of subcellular compartments termed membraneless organelles. The field has made great strides in elucidating the molecular basis of biomolecular phase separation in various disease, stress-response and developmental contexts. Many important biological consequences of such “condensation” are now emerging from in vivo studies. Here we review recent work from our group and others showing that many proteins undergo rapid, reversible condensation in the cellular response to ubiquitous environmental fluctuations such as osmotic changes. We discuss molecular crowding as an important driver of condensation in these responses and suggest that a significant fraction of the proteome is poised to undergo phase separation under physiological conditions. In addition, we review methods currently emerging to visualize, quantify and modulate the dynamics of intracellular condensates in live cells. Finally, we propose a metaphor for rapid phase separation based on cloud formation, reasoning that our familiar experiences with the readily reversible condensation of water droplets help understand the principle of phase separation. Overall, we provide an account of how biological phase separation supports the highly intertwined relationship between the composition and dynamic internal organization of cells, thus facilitating extremely rapid reorganization in response to internal and external fluctuations.

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“…4. Single particle tracking (SPT) is a powerful tool to study dynamic recruitment of molecules to condensates ( 45 , 65 , 136 ). 5.…”

Section: Physicochemical Underpinnings Of Phase Separationmentioning

confidence: 99%

Hyperosmotic phase separation: Condensates beyond inclusions, granules and organelles

Jalihal

Schmidt

Gao

et al. 2021

Journal of Biological Chemistry

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“…If donor and acceptor fluorescence are simultaneously monitored, the method is very sensitive (Joo, Balci, Ishitsuka, Buranachai, & Ha, 2008). For this reason FRET is the method of choice to measure single molecule fluorescence (Jalihal, Lund, & Walter, 2019;Ray, Widom, & Walter, 2018;R. Roy, Hohng, & Ha, 2008).…”

Section: Fluorescence Resonance Energy Transfermentioning

confidence: 99%

“…Single molecule methods have provided insight into biological systems beyond the scope of ensemble methods, and have transformed the quantitative understanding of complex reaction kinetics, such as translation and pre-mRNA splicing (Ray et al, 2018;van der Feltz & Hoskins, 2017). Single molecule techniques have also added critical new ways to characterize RNA helicases (Dulin, Berghuis, Depken, & Dekker, 2015;Konig, Liyanage, Sigel, & Rueda, 2013) and RNA-binding proteins (Jalihal et al, 2019).…”

Section: Single Molecule Approaches To Measure the Kinetics Of Rna-protein Interactionsmentioning

confidence: 99%

Approaches for measuring the dynamics of RNA–protein interactions

Licatalosi

Jankowsky

2019

WIREs RNA

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RNA-protein interactions are pivotal for the regulation of gene expression from bacteria to human. RNA-protein interactions are dynamic; they change over biologically relevant timescales. Understanding the regulation of gene expression at the RNA level therefore requires knowledge of the dynamics of RNA-protein interactions. Here, we discuss the main experimental approaches to measure dynamic aspects of RNA-protein interactions. We cover techniques that assess dynamics of cellular RNA-protein interactions that accompany biological processes over timescales of hours or longer and techniques measuring the kinetic dynamics of RNA-protein interactions in vitro.

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“…smFISH provides a snapshot of the subcellular, spatial distribution of RNAs at single molecule resolution in fixed cells (Femino, Fay, Fogarty, & Singer, 1998;George, Indig, Abdelmohsen, & Gorospe, 2018;Pitchiaya et al, 2014;Tutucci, Livingston, Singer, & Wu, 2018). With advances in fluorescence detection hardware and the development of protein-based and chemical probes that allow powerful labeling of mRNAs, several single-color single molecule techniques have been developed to track diverse classes of RNAs in living cells and tissues (Bertrand et al, 1998;Jalihal, Lund, & Walter, 2019;Lim & Peabody, 2002;Lyon & Stasevich, 2017;Pitchiaya et al, 2014;Tutucci, Livingston, et al, 2018;Tutucci, Vera, et al, 2018). These methods have provided significant insights into the dynamic and stochastic nature of gene expression at the single molecule level that leads to a high degree of heterogeneity among individual cells.…”

Section: Introductionmentioning

confidence: 99%

Following the messenger: Recent innovations in live cell single molecule fluorescence imaging

et al. 2020

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Messenger RNAs (mRNAs) convey genetic information from the DNA genome to proteins and thus lie at the heart of gene expression and regulation of all cellular activities. Live cell single molecule tracking tools enable the investigation of mRNA trafficking, translation and degradation within the complex environment of the cell and in real time. Over the last 5 years, nearly all tools within the mRNA tracking toolbox have been improved to achieve high‐quality multi‐color tracking in live cells. For example, the bacteriophage‐derived MS2‐MCP system has been improved to facilitate cloning and achieve better signal‐to‐noise ratio, while the newer PP7‐PCP system now allows for orthogonal tracking of a second mRNA or mRNA region. The coming of age of epitope‐tagging technologies, such as the SunTag, MoonTag and Frankenbody, enables monitoring the translation of single mRNA molecules. Furthermore, the portfolio of fluorogenic RNA aptamers has been expanded to improve cellular stability and achieve a higher fluorescence “turn‐on” signal upon fluorogen binding. Finally, microinjection‐based tools have been shown to be able to track multiple RNAs with only small fluorescent appendages and to track mRNAs together with their interacting partners. We systematically review and compare the advantages, disadvantages and demonstrated applications in discovering new RNA biology of this refined, expanding toolbox. Finally, we discuss developments expected in the near future based on the limitations of the current methods. This article is categorized under: RNA Export and Localization > RNA Localization RNA Structure and Dynamics > RNA Structure, Dynamics, and Chemistry RNA Interactions with Proteins and Other Molecules > RNA–Protein Complexes

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Coming Together: RNAs and Proteins Assemble under the Single-Molecule Fluorescence Microscope

Cited by 12 publications

References 116 publications

Hyperosmotic phase separation: Condensates beyond inclusions, granules and organelles

Hyperosmotic phase separation: Condensates beyond inclusions, granules and organelles

Approaches for measuring the dynamics of RNA–protein interactions

Following the messenger: Recent innovations in live cell single molecule fluorescence imaging

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