Coexisting Liquid Phases Underlie Nucleolar Subcompartments

doi:10.1016/J.CELL.2016.04.047

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Coexisting Liquid Phases Underlie Nucleolar Subcompartments

Journal Article•DOI•

Coexisting Liquid Phases Underlie Nucleolar Subcompartments

Marina Feric¹, Nilesh Vaidya¹, Tyler S. Harmon², Diana M. Mitrea³, Lian Zhu¹, Tiffany M. Richardson¹, Richard W. Kriwacki³, Rohit V. Pappu², Clifford P. Brangwynne¹ - Show less +5 more•Institutions (3)

Princeton University¹, Washington University in St. Louis², St. Jude Children's Research Hospital³

16 Jun 2016-Cell (Elsevier)-Vol. 165, Iss: 7, pp 1686-1697

TL;DR: It is shown that subcompartments within the nucleolus represent distinct, coexisting liquid phases that may facilitate sequential RNA processing reactions in a variety of RNP bodies, and suggested that phase separation can give rise to multilayered liquids.

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About: This article is published in Cell.The article was published on 2016-06-16 and is currently open access. It has received 1330 citations till now.

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Journal Article•DOI•

Biomolecular condensates: organizers of cellular biochemistry

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Salman F. Banani¹, Hyun-Ok Kate Lee², Anthony A. Hyman², Michael K. Rosen¹•Institutions (2)

University of Texas Southwestern Medical Center¹, Max Planck Society²

01 May 2017-Nature Reviews Molecular Cell Biology

TL;DR: This work has shown that liquid–liquid phase separation driven by multivalent macromolecular interactions is an important organizing principle for biomolecular condensates and has proposed a physical framework for this organizing principle.

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Abstract: In addition to membrane-bound organelles, eukaryotic cells feature various membraneless compartments, including the centrosome, the nucleolus and various granules. Many of these compartments form through liquid–liquid phase separation, and the principles, mechanisms and regulation of their assembly as well as their cellular functions are now beginning to emerge. Biomolecular condensates are micron-scale compartments in eukaryotic cells that lack surrounding membranes but function to concentrate proteins and nucleic acids. These condensates are involved in diverse processes, including RNA metabolism, ribosome biogenesis, the DNA damage response and signal transduction. Recent studies have shown that liquid–liquid phase separation driven by multivalent macromolecular interactions is an important organizing principle for biomolecular condensates. With this physical framework, it is now possible to explain how the assembly, composition, physical properties and biochemical and cellular functions of these important structures are regulated.

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3,294 citations

Journal Article•DOI•

Liquid phase condensation in cell physiology and disease.

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Yongdae Shin¹, Clifford P. Brangwynne¹•Institutions (1)

Princeton University¹

22 Sep 2017-Science

TL;DR: The findings together suggest that several membrane-less organelles have been shown to exhibit a concentration threshold for assembly, a hallmark of phase separation, and represent liquid-phase condensates, which form via a biologically regulated (liquid-liquid) phase separation process.

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Abstract: BACKGROUND Living cells contain distinct subcompartments to facilitate spatiotemporal regulation of biological reactions. In addition to canonical membrane-bound organelles such as secretory vesicles and endoplasmic reticulum, there are many organelles that do not have an enclosing membrane yet remain coherent structures that can compartmentalize and concentrate specific sets of molecules. Examples include assemblies in the nucleus such as the nucleolus, Cajal bodies, and nuclear speckles and also cytoplasmic structures such as stress granules, P-bodies, and germ granules. These structures play diverse roles in various biological processes and are also increasingly implicated in protein aggregation diseases. ADVANCES A number of studies have shown that membrane-less assemblies exhibit remarkable liquid-like features. As with conventional liquids, they typically adopt round morphologies and coalesce into a single droplet upon contact with one another and also wet intracellular surfaces such as the nuclear envelope. Moreover, component molecules exhibit dynamic exchange with the surrounding nucleoplasm and cytoplasm. These findings together suggest that these structures represent liquid-phase condensates, which form via a biologically regulated (liquid-liquid) phase separation process. Liquid phase condensation increasingly appears to be a fundamental mechanism for organizing intracellular space. Consistent with this concept, several membrane-less organelles have been shown to exhibit a concentration threshold for assembly, a hallmark of phase separation. At the molecular level, weak, transient interactions between molecules with multivalent domains or intrinsically disordered regions (IDRs) are a driving force for phase separation. In cells, condensation of liquid-phase assemblies can be regulated by active processes, including transcription and various posttranslational modifications. The simplest physical picture of a homogeneous liquid phase is often not enough to capture the full complexity of intracellular condensates, which frequently exhibit heterogeneous multilayered structures with partially solid-like characters. However, recent studies have shown that multiple distinct liquid phases can coexist and give rise to richly structured droplet architectures determined by the relative liquid surface tensions. Moreover, solid-like phases can emerge from metastable liquid condensates via multiple routes of potentially both kinetic and thermodynamic origins, which has important implications for the role of intracellular liquids in protein aggregation pathologies. OUTLOOK The list of intracellular assemblies driven by liquid phase condensation is growing rapidly, but our understanding of their sequence-encoded biological function and dysfunction lags behind. Moreover, unlike equilibrium phases of nonliving matter, living cells are far from equilibrium, with intracellular condensates subject to various posttranslational regulation and other adenosine triphosphate–dependent biological activity. Efforts using in vitro reconstitution, combined with traditional cell biology approaches and quantitative biophysical tools, are required to elucidate how such nonequilibrium features of living cells control intracellular phase behavior. The functional consequences of forming liquid condensates are likely multifaceted and may include facilitated reaction, sequestration of specific factors, and organization of associated intracellular structures. Liquid phase condensation is particularly interesting in the nucleus, given the growing interest in the impact of nuclear phase behavior on the flow of genetic information; nuclear condensates range from micrometer-sized bodies such as the nucleolus to submicrometer structures such as transcriptional assemblies, all of which directly interact with and regulate the genome. Deepening our understanding of these intracellular states of matter not only will shed light on the basic biology of cellular organization but also may enable therapeutic intervention in protein aggregation disease by targeting intracellular phase behavior.

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2,432 citations

Cites background from "Coexisting Liquid Phases Underlie N..."

...Remarkably, when these two different types of purified protein droplets are combined, they do not fuse with one another but remain as distinct, immiscible phases (65)....
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...Consistent with this, the viscoelasticity of nucleoli, particularly the fibrillarin-rich nucleolar subcompartment, exhibits a strong ATP dependence (13, 65), whereas stress granule component dynamics decrease upon ATP depletion (62)....
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...liquid phase nature of P granules (12), nucleoli (13, 64, 65), and dozens of other such fluid...
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Journal Article•DOI•

Coactivator condensation at super-enhancers links phase separation and gene control

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Benjamin R. Sabari¹, Alessandra Dall’Agnese¹, Ann Boija¹, Isaac A. Klein², Isaac A. Klein¹, Eliot L. Coffey¹, Krishna Shrinivas¹, Brian J. Abraham¹, Nancy M. Hannett¹, Alicia V. Zamudio¹, John C. Manteiga¹, Charles H. Li¹, Yang Eric Guo¹, Daniel S. Day¹, Jurian Schuijers¹, Eliza Vasile¹, Sohail Malik³, Denes Hnisz¹, Tong Ihn Lee¹, Ibrahim I Cisse¹, Robert G. Roeder³, Phillip A. Sharp¹, Arup K. Chakraborty, Richard A. Young¹ - Show less +20 more•Institutions (3)

Massachusetts Institute of Technology¹, Harvard University², Rockefeller University³

27 Jul 2018-Science

TL;DR: It is postulated that super-enhancers are phase-separated multimolecular assemblies, also known as biomolecular condensates, which provide a means to compartmentalize and concentrate biochemical reactions within cells.

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Abstract: Super-enhancers (SEs) are clusters of enhancers that cooperatively assemble a high density of transcriptional apparatus to drive robust expression of genes with prominent roles in cell identity. Here, we demonstrate that the SE-enriched transcriptional coactivators BRD4 and MED1 form nuclear puncta at SEs that exhibit properties of liquid-like condensates and are disrupted by chemicals that perturb condensates. The intrinsically disordered regions (IDRs) of BRD4 and MED1 can form phase-separated droplets and MED1-IDR droplets can compartmentalize and concentrate transcription apparatus from nuclear extracts. These results support the idea that coactivators form phase-separated condensates at SEs that compartmentalize and concentrate the transcription apparatus, suggest a role for coactivator IDRs in this process, and offer insights into mechanisms involved in control of key cell identity genes.

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1,506 citations

Journal Article•DOI•

Considerations and Challenges in Studying Liquid-Liquid Phase Separation and Biomolecular Condensates.

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Simon Alberti¹, Simon Alberti², Amy S. Gladfelter³, Amy S. Gladfelter⁴, Tanja Mittag⁵ - Show less +1 more•Institutions (5)

Max Planck Society¹, Dresden University of Technology², Marine Biological Laboratory³, University of North Carolina at Chapel Hill⁴, St. Jude Children's Research Hospital⁵

24 Jan 2019-Cell

TL;DR: In this article, the authors propose guidelines for rigorous experimental characterization of liquid-liquid phase separation processes in vitro and in cells, discuss the caveats of common experimental approaches, and point out experimental and theoretical gaps in the field.

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1,482 citations

Cites background from "Coexisting Liquid Phases Underlie N..."

...Coarse-grained simulations of multi-component systems have recently provided insight into the physical principles that may drive the formation of multilayered membraneless organelles such as nucleoli and nuclear speckles (Fei et al., 2017; Feric et al., 2016)....
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...Lastly, returning to one of the earliest identified membraneless organelles, there is growing evidence that the liquid-like state of the nucleolusmay be important for the assembly of ribosomes (Feric et al., 2016; Lee et al., 2016; Mitrea et al., 2016)....
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...The rules by which molecules are sorted into different coexisting condensates are largely unclear, although a role for sequence features in RNAs and proteins and relative surface tension has been revealed (Feric et al., 2016; Langdon et al., 2018)....
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...Measurement of contact angles (the angle between the coverglass and the droplet surface) can also provide important information on surface tension and the chemical nature of the surface of droplets (Feric et al., 2016)....
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...The combination of favorable and unfavorable interactions between different components resulted in the non-random organization that was observed experimentally (Fei et al., 2017; Feric et al., 2016)....
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Journal Article•DOI•

Protein Phase Separation: A New Phase in Cell Biology.

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Steven Boeynaems¹, Steven Boeynaems², Simon Alberti³, Nicolas L. Fawzi⁴, Tanja Mittag⁵, Magdalini Polymenidou⁶, Frederic Rousseau⁷, Frederic Rousseau², Joost Schymkowitz⁷, Joost Schymkowitz², James Shorter⁸, Benjamin Wolozin⁹, Ludo Van Den Bosch², Peter Tompa¹⁰, Peter Tompa¹¹, Monika Fuxreiter¹² - Show less +12 more•Institutions (12)

Stanford University¹, Katholieke Universiteit Leuven², Max Planck Society³, Brown University⁴, St. Jude Children's Research Hospital⁵, University of Zurich⁶, Switch⁷, University of Pennsylvania⁸, Boston University⁹, Hungarian Academy of Sciences¹⁰, Vrije Universiteit Brussel¹¹, University of Debrecen¹²

01 Jun 2018-Trends in Cell Biology

TL;DR: A combination of techniques from cell biology, biophysics, physical chemistry, structural biology, and bioinformatics are starting to help establish the molecular principles of an emerging field, thus paving the way for exciting discoveries, including novel therapeutic approaches for the treatment of age-related disorders.

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1,317 citations

Cites background from "Coexisting Liquid Phases Underlie N..."

...These results point to a complex internal organization of SGs, a picture that, given recent work, is likely true for other membraneless organelles as well [24,25,68]....
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..., in the nucleolus; Figure 1D)? How can amultiphase system such as the nucleolus be assembled and controlled? Recent work has suggested that differences in surface tension of protein droplets could mediate the formation of such multiphase droplets [24]....
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References

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Journal Article•DOI•

Germline P Granules Are Liquid Droplets That Localize by Controlled Dissolution/Condensation

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Clifford P. Brangwynne¹, Christian R. Eckmann¹, David S. Courson², Agata Rybarska¹, Carsten Hoege¹, Jöbin Gharakhani¹, Jöbin Gharakhani², Frank Jülicher¹, Frank Jülicher², Anthony A. Hyman², Anthony A. Hyman¹ - Show less +7 more•Institutions (2)

Max Planck Society¹, Marine Biological Laboratory²

26 Jun 2009-Science

TL;DR: It is shown that P granules exhibit liquid-like behaviors, including fusion, dripping, and wetting, which is used to estimate their viscosity and surface tension, and reflects a classic phase transition, in which polarity proteins vary the condensation point across the cell.

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Abstract: In sexually reproducing organisms, embryos specify germ cells, which ultimately generate sperm and eggs In Caenorhabditis elegans, the first germ cell is established when RNA and protein-rich P granules localize to the posterior of the one-cell embryo Localization of P granules and their physical nature remain poorly understood Here we show that P granules exhibit liquid-like behaviors, including fusion, dripping, and wetting, which we used to estimate their viscosity and surface tension As with other liquids, P granules rapidly dissolved and condensed Localization occurred by a biased increase in P granule condensation at the posterior This process reflects a classic phase transition, in which polarity proteins vary the condensation point across the cell Such phase transitions may represent a fundamental physicochemical mechanism for structuring the cytoplasm

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2,134 citations

"Coexisting Liquid Phases Underlie N..." refers background in this paper

...Many of these RNP bodies exhibit liquid-like biophysical properties, and growing evidence suggests they assemble via liquidliquid phase separation (Brangwynne et al., 2009; Nott et al., 2015; Li et al., 2012; Weber and Brangwynne, 2015)....
[...]

Journal Article•DOI•

A Liquid-to-Solid Phase Transition of the ALS Protein FUS Accelerated by Disease Mutation

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Avinash Patel¹, Hyun O. Lee¹, Louise Jawerth¹, Shovamayee Maharana¹, Marcus Jahnel¹, Marco Y. Hein¹, Stoyno S. Stoynov², Julia Mahamid¹, Shambaditya Saha¹, Titus M. Franzmann¹, Andrej Pozniakovski¹, Ina Poser¹, Nicola Maghelli¹, Loic Royer¹, Martin Weigert¹, Eugene W. Myers¹, Stephan W. Grill¹, David N. Drechsel¹, Anthony A. Hyman¹, Simon Alberti¹ - Show less +16 more•Institutions (2)

Max Planck Society¹, Bulgarian Academy of Sciences²

27 Aug 2015-Cell

TL;DR: It is proposed that liquid-like compartments carry the trade-off between functionality and risk of aggregation and that aberrant phase transitions within liquid- like compartments lie at the heart of ALS and, presumably, other age-related diseases.

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1,988 citations

"Coexisting Liquid Phases Underlie N..." refers background in this paper

...…(IDP/LCS) drive phase transitions underlying assembly of the nucleolus (Berry et al., 2015), stress granules (Wippich et al., 2013; Molliex et al., 2015; Patel et al., 2015), P granules and nuage bodies (Elbaum-Garfinkle et al., 2015; Wang et al., 2014), and nuclear speckles (Hennig et al., 2015)....
[...]
...…disordered regions can facilitate the transition from liquid-like to solid-like structures (Weber and Brangwynne, 2012), which is supported by recent studies on a variety of RNA binding proteins (Zhang et al., 2015; Patel et al., 2015; Lin et al., 2015; Molliex et al., 2015; Xiang et al., 2015)....
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Journal Article•DOI•

Phase Separation by Low Complexity Domains Promotes Stress Granule Assembly and Drives Pathological Fibrillization

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Amandine Molliex¹, Jamshid Temirov¹, Jihun Lee¹, Maura Coughlin¹, Anderson P. Kanagaraj¹, Hong Joo Kim¹, Tanja Mittag¹, J. Paul Taylor¹ - Show less +4 more•Institutions (1)

St. Jude Children's Research Hospital¹

24 Sep 2015-Cell

TL;DR: It is demonstrated that the disease-related RBP hnRNPA1 undergoes liquid-liquid phase separation (LLPS) into protein-rich droplets mediated by a low complexity sequence domain (LCD), and suggested that LCD-mediated LLPS contributes to the assembly of stress granules and their liquid properties.

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1,947 citations

"Coexisting Liquid Phases Underlie N..." refers background or result in this paper

...…sequences (IDP/LCS) drive phase transitions underlying assembly of the nucleolus (Berry et al., 2015), stress granules (Wippich et al., 2013; Molliex et al., 2015; Patel et al., 2015), P granules and nuage bodies (Elbaum-Garfinkle et al., 2015; Wang et al., 2014), and nuclear speckles…...
[...]
...…disordered regions can facilitate the transition from liquid-like to solid-like structures (Weber and Brangwynne, 2012), which is supported by recent studies on a variety of RNA binding proteins (Zhang et al., 2015; Patel et al., 2015; Lin et al., 2015; Molliex et al., 2015; Xiang et al., 2015)....
[...]
..., 2015), stress granules (Wippich et al., 2013; Molliex et al., 2015; Patel et al., 2015), P granules and nuage bodies (Elbaum-Garfinkle et al....
[...]
...These data are consistent with the hypothesis that disordered regions can facilitate the transition from liquid-like to solid-like structures (Weber and Brangwynne, 2012), which is supported by recent studies on a variety of RNA binding proteins (Zhang et al., 2015; Patel et al., 2015; Lin et al., 2015; Molliex et al., 2015; Xiang et al., 2015)....
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Journal Article•DOI•

Phase transitions in the assembly of multivalent signalling proteins

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Pilong Li¹, Sudeep Banjade¹, Hui-Chun Cheng¹, Soyeon Kim¹, Baoyu Chen¹, Liang Guo², Marc C. Llaguno¹, Javoris Hollingsworth³, David S. King⁴, Salman F. Banani¹, Paul S. Russo³, Qiu-Xing Jiang, B. Tracy Nixon⁵, Michael K. Rosen - Show less +10 more•Institutions (5)

University of Texas Southwestern Medical Center¹, Argonne National Laboratory², Louisiana State University³, University of California, Berkeley⁴, Pennsylvania State University⁵

15 Mar 2012-Nature

TL;DR: Interactions between diverse synthetic, multivalent macromolecules (including multi-domain proteins and RNA) produce sharp liquid–liquid-demixing phase separations, generating micrometre-sized liquid droplets in aqueous solution.

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Abstract: Cells are organized on length scales ranging from angstrom to micrometres. However, the mechanisms by which angstrom-scale molecular properties are translated to micrometre-scale macroscopic properties are not well understood. Here we show that interactions between diverse synthetic, multivalent macromolecules (including multi-domain proteins and RNA) produce sharp liquid-liquid-demixing phase separations, generating micrometre-sized liquid droplets in aqueous solution. This macroscopic transition corresponds to a molecular transition between small complexes and large, dynamic supramolecular polymers. The concentrations needed for phase transition are directly related to the valency of the interacting species. In the case of the actin-regulatory protein called neural Wiskott-Aldrich syndrome protein (N-WASP) interacting with its established biological partners NCK and phosphorylated nephrin, the phase transition corresponds to a sharp increase in activity towards an actin nucleation factor, the Arp2/3 complex. The transition is governed by the degree of phosphorylation of nephrin, explaining how this property of the system can be controlled to regulatory effect by kinases. The widespread occurrence of multivalent systems suggests that phase transitions may be used to spatially organize and biochemically regulate information throughout biology.

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1,816 citations

"Coexisting Liquid Phases Underlie N..." refers background in this paper

...Many of these RNP bodies exhibit liquid-like biophysical properties, and growing evidence suggests they assemble via liquidliquid phase separation (Brangwynne et al., 2009; Nott et al., 2015; Li et al., 2012; Weber and Brangwynne, 2015)....
[...]

Journal Article•DOI•

Stress granules and processing bodies are dynamically linked sites of mRNP remodeling

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Nancy Kedersha¹, Georg Stoecklin¹, Maranatha Ayodele¹, Patrick W. Yacono¹, Jens Lykke-Andersen², Marvin J. Fritzler³, Donalyn Scheuner⁴, Randal J. Kaufman⁴, David E. Golan¹, Paul A. Anderson¹ - Show less +6 more•Institutions (4)

Brigham and Women's Hospital¹, University of Colorado Boulder², University of Calgary³, University of Michigan⁴

20 Jun 2005-Journal of Cell Biology

TL;DR: It is proposed that mRNA released from disassembled polysomes is sorted and remodeled at SGs, from which selected transcripts are delivered to PBs for degradation, an interaction that is promoted by the related mRNA decay factors TTP and BRF1.

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Abstract: Stress granules (SGs) are cytoplasmic aggregates of stalled translational preinitiation complexes that accumulate during stress. GW bodies/processing bodies (PBs) are distinct cytoplasmic sites of mRNA degradation. In this study, we show that SGs and PBs are spatially, compositionally, and functionally linked. SGs and PBs are induced by stress, but SG assembly requires eIF2alpha phosphorylation, whereas PB assembly does not. They are also dispersed by inhibitors of translational elongation and share several protein components, including Fas-activated serine/threonine phosphoprotein, XRN1, eIF4E, and tristetraprolin (TTP). In contrast, eIF3, G3BP, eIF4G, and PABP-1 are restricted to SGs, whereas DCP1a and 2 are confined to PBs. SGs and PBs also can harbor the same species of mRNA and physically associate with one another in vivo, an interaction that is promoted by the related mRNA decay factors TTP and BRF1. We propose that mRNA released from disassembled polysomes is sorted and remodeled at SGs, from which selected transcripts are delivered to PBs for degradation.

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1,366 citations