DOCSLIB.ORG

Enhancers and Phase Separation in the Control of Gene Expression

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
Enhancers and Phase Separation in the Control of Gene Expression Enhancers and phase separation in the control of gene expression By John C. Manteiga B.S., University of Massachusetts Amherst (2011) Submitted to the Department of Biology in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy at the Massachusetts Institute of Technology February 2020 © 2020 Massachusetts Institute of Technology. All rights reserved. The author hereby grants to MIT permission to reproduce or distribute publicly paper and electronic copies of this thesis document in whole or in part in any medium now known or hereafter created. Signature of Author:____________________________________________________________ Department of Biology October 21st, 2019 Certified by:__________________________________________________________________ Richard A. Young Member, Whitehead Institute Professor of Biology Thesis Supervisor Accepted by:__________________________________________________________________ Steve Bell Professor of Biology Co-Chair, Biology Graduate Committee 2 Enhancers and phase separation in the control of gene expression By John C. Manteiga Submitted to the Department of Biology on November 15th, 2019 in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy at the Massachusetts Institute of Technology ABSTRACT Gene regulation underlies the control of cell identity, development, and disease. Transcription of genes is regulated by DNA elements called enhancers, which are bound by transcription factors and coactivators, leading to the recruitment of RNA polymerase II and the production of RNA. Enhancers are thought to loop to specific gene promoters to stimulate transcription, but the mechanisms that cause enhancers to selectively loop to specific gene promoters is not well understood. In this thesis, I first describe new insights into enhancer-promoter loop specificity from studies examining the mechanisms that allow tumor-specific super-enhancers to loop to the MYC oncogene in diverse cancer types (Schuijers and Manteiga et al., 2018). While conducting these studies, it was proposed that super-enhancers and the factors associated with them form liquid-liquid phase-separated condensates. Following this proposal, I contributed to collaborative studies that strongly supported this model (Boija et al., 2018; Sabari et al., 2018, see Appendix I and II of this thesis). This model of transcription led me to ask how key transcriptional components could be recruited into super-enhancer condensates. I performed studies showing that the interaction of RNA polymerase II with these condensates involves the large heptapeptide repeat of the C-terminal domain (CTD) of the enzyme. Furthermore, these studies provided evidence that phosphorylation of the CTD, which is associated with the initiation to elongation transition, weakens these interactions, thus facilitating the transition of RNA polymerase II into different condensates involved in co-transcriptional splicing of the nascent transcript (Guo and Manteiga et al., 2019). These studies provide new insights into the mechanisms of enhancer-promoter interaction, roles for the RNA polymerase II CTD in the enzyme’s partitioning into nuclear condensates, and a role for phosphorylation in switching the nuclear condensate partitioning behavior of RNA polymerase II. Thesis supervisor: Richard A. Young Title: Member, Whitehead Institute; Professor of Biology 3 4 ACKNOWLEDGEMENTS Many people shaped my experiences while at MIT and deserve great thanks. Beginning on the scientific side, I am extremely grateful to all the members of the Young lab. I learned so much from being part of this group and you all made coming to work every day worth it. I’d like to give Jurian Schuijers a special thanks for taking me under his wing when I first joined the lab. Alicia, Jon, Ben, Abe, and many others were also great friends and scientists. Rick deserves a special thanks for taking me into the lab, and for creating an environment where incredible research can happen. I learned so much about how to plan, execute, and communicate science under Rick’s guidance. I am also grateful to my thesis committee of Phil Sharp and Laurie Boyer, who always asked the right questions and helped keep me on the right course. There were also many people outside of MIT who supported me along the way. To my friends: Lucas, Nick, Jason, the North Andover gang, the UMass crew, the Boston Spikeball group, I want to say thanks for the fun times and sometimes greatly needed distraction from research. Of course, none of this would have been possible without my loving family: Mom, Dad, Carrie, and Matt. Finally, I want to thank my wonderful fiancé Aly. From meeting at UMass to moving to Boston for our PhDs, Aly has always been by my side tackling life’s challenges with me. She makes every day better and I am so glad we get to get married this year. 5 STATEMENT ON WORK PRESENTED Chapter 1 I wrote Chapter 1 with input and minor edits from Rick Young. Chapter 2 I performed experiments and analyses in Figures 2 and 4 and contributed to the design of the others. Jurian Schuijers and Rick Young wrote the manuscript with input from me and the other authors. Chapter 3 I either performed or was directly involved in the design of all experiments and analyses. Rick Young, myself, and Eric Guo wrote the manuscript with input from the other authors. Chapter 4 I wrote Chapter 4 with input and minor edits from Rick Young. Appendix I I was heavily involved in the development of the analyses performed on the immunofluorescence data and provided input in the writing of the manuscript. Appendix II I performed a subset of the immunofluorescence experiments and provided input in the writing of the manuscript. 6 TABLE OF CONTENTS ABSTRACT ............................................................................................................................... 3 ACKNOWLEDGEMENTS .......................................................................................................... 5 STATEMENT ON WORK PRESENTED .................................................................................... 6 CHAPTER 1: INTRODUCTION ................................................................................................. 9 Overview .................................................................................................................................................. 9 Transcriptional control of gene expression and enhancers ............................................................ 11 Condensates compartmentalize cellular biochemistry .................................................................... 14 Super-enhancers form condensates .................................................................................................. 18 Co-transcriptional splicing may occur in splicing factor condensates ......................................... 22 References ............................................................................................................................................. 25 CHAPTER 2: TRANSCRIPTIONAL DYSREGULATION OF MYC REVEALS COMMON ENHANCER-DOCKING MECHANISM .....................................................................................33 Summary ................................................................................................................................................ 34 Introduciton ........................................................................................................................................... 35 Results ................................................................................................................................................... 36 Discussion ............................................................................................................................................. 42 References ............................................................................................................................................. 45 Experimental procedures ..................................................................................................................... 51 Supplemental figures ........................................................................................................................... 53 CHAPTER 3: POL II PHOSPHORYLATION REGULATES A SWITCH BETWEEN TRANSCRIPTIONAL AND SPLICING CONDENSATES .........................................................63 Summary ................................................................................................................................................ 64 Main Text ............................................................................................................................................... 64 References ............................................................................................................................................. 72 Methods ................................................................................................................................................. 74 Extended data ....................................................................................................................................... 85 Supplemental figures ........................................................................................................................... 94 CHAPTER 4: FUTURE DIRECTIONS AND DISCUSSION .......................................................96 The molecular grammar of phase separation ...................................................................................
Load more

Recommended publications
  • Liquid Network Connectivity Regulates the Stability and Composition Of
    Liquid network connectivity regulates the stability and composition of biomolecular condensates with many components Jorge R. Espinosaa,b,c, Jerelle A. Josepha,b,c , Ignacio Sanchez-Burgosa,b,c , Adiran Garaizara,b,c , Daan Frenkelb , and Rosana Collepardo-Guevaraa,b,c,1 aMaxwell Centre, Cavendish Laboratory, Department of Physics, University of Cambridge, Cambridge CB3 0HE, United Kindgdom; bDepartment of Chemistry, University of Cambridge, Cambridge CB2 1EW, United Kingdom; and cDepartment of Genetics, University of Cambridge, Cambridge CB2 3EH, United Kingdom Edited by Ken A. Dill, Stony Brook University, Stony Brook, NY, and approved April 17, 2020 (received for review October 8, 2019) One of the key mechanisms used by cells to control the spatiotem- 10), cellular bodies (11), or otherwise (8)—are ubiquitous within poral organization of their many components is the formation and the cell interior. Biomolecular condensates account for numer- dissolution of biomolecular condensates through liquid–liquid ous highly diverse domains, both inside the cytoplasm [e.g., P phase separation (LLPS). Using a minimal coarse-grained model granules (12) and RNA granules/bodies (13–15)] and in the cell that allows us to simulate thousands of interacting multivalent nucleus [e.g., Cajal bodies (16), nucleoli (17), heterochromatin proteins, we investigate the physical parameters dictating the domains (18, 19), the transport channels in the nuclear pore stability and composition of multicomponent biomolecular con- complex (20), and possibly superenhancers
    [Show full text]
  • S41598-020-74080-2.Pdf
    www.nature.com/scientificreports OPEN Multivalent tumor suppressor adenomatous polyposis coli promotes Axin biomolecular condensate formation and efcient β‑catenin degradation Tie‑Mei Li1,2,5*, Jing Ren3, Dylan Husmann2, John P. Coan1,2, Or Gozani2* & Katrin F. Chua1,4* The tumor suppressor adenomatous polyposis coli (APC) is frequently mutated in colorectal cancers. APC and Axin are core components of a destruction complex that scafolds GSK3β and CK1 to earmark β‑catenin for proteosomal degradation. Disruption of APC results in pathologic stabilization of β‑catenin and oncogenesis. However, the molecular mechanism by which APC promotes β‑catenin degradation is unclear. Here, we fnd that the intrinsically disordered region (IDR) of APC, which contains multiple β‑catenin and Axin interacting sites, undergoes liquid–liquid phase separation (LLPS) in vitro. Expression of the APC IDR in colorectal cells promotes Axin puncta formation and β‑catenin degradation. Our results support the model that multivalent interactions between APC and Axin drives the β‑catenin destruction complex to form biomolecular condensates in cells, which concentrate key components to achieve high efcient degradation of β‑catenin. APC mutations are present in ~ 80% of colorectal cancer cases1, and typically cause truncation of the APC pro- tein. APC functions downstream of the Wnt signalosome, and it is essential for the degradation of β-catenin in the absence of Wnt stimulation 2. APC forms a complex, termed the “β-catenin destruction complex” or “Axin degradasome” composed of β-catenin, the scafold protein Axin, and two kinases: GSK3β and casein kinase 1 (CK1)3,4. In the complex, proximity of β-catenin to the two kinases leads to β-catenin phosphorylation, which in turn facilitates its ubiquitination and proteosomal degradation.
    [Show full text]
  • Structure of Biomolecular Condensates from Dissipative Particle Dynamics Simulations
    bioRxiv preprint doi: https://doi.org/10.1101/2019.12.11.873133; this version posted April 30, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. Structure of biomolecular condensates from dissipative particle dynamics simulations Julian C. Shillcock,1* Maelick Brochut,2 Etienne Chénais,2 and John H. Ipsen3 1 Laboratory of Molecular and Chemical Biology of Neurodegeneration, Ecole polytechnique fédérale de Lausanne, CH-1015 Lausanne, Switzerland 2 Brain Mind Institute, Ecole polytechnique fédérale de Lausanne, CH-1015 Lausanne, Switzerland 3 Dept. of Physics, Chemistry and Pharmacy, University of Southern Denmark, Campusvej 55, DK-5230 Odense M, Denmark * Correspondence: [email protected], OrcId: 0000-0002-7885-735X ABSTRACT Phase separation of immiscible fluids is a common phenomenon in polymer chemistry, and is recognized as an important mechanism by which cells compartmentalize their biochemical reactions. Biomolecular condensates are condensed fluid droplets in cells that form by liquid-liquid phase separation of intrinsically-disordered proteins. They have a wide range of functions and are associated with chronic neurodegenerative diseases in which they become pathologically rigid. Intrinsically-disordered proteins are conformationally flexible and possess multiple, distributed binding sites for each other or for RNA. However, it remains unclear how their material properties depend on the molecular structure of the proteins. Here we use coarse-grained simulations to explore the phase behavior and structure of a model biomolecular condensate composed of semi-flexible polymers with attractive end-caps in a good solvent. Although highly simplified, the model contains the minimal molecular features that are sufficient to observe liquid-liquid phase separation of soluble polymers.
    [Show full text]
  • The Nucleolus As a Multiphase Liquid Condensate
    REVIEWS The nucleolus as a multiphase liquid condensate Denis L. J. Lafontaine 1 ✉ , Joshua A. Riback 2, Rümeyza Bascetin 1 and Clifford P. Brangwynne 2,3 ✉ Abstract | The nucleolus is the most prominent nuclear body and serves a fundamentally important biological role as a site of ribonucleoprotein particle assembly, primarily dedicated to ribosome biogenesis. Despite being one of the first intracellular structures visualized historically, the biophysical rules governing its assembly and function are only starting to become clear. Recent studies have provided increasing support for the concept that the nucleolus represents a multilayered biomolecular condensate, whose formation by liquid–liquid phase separation (LLPS) facilitates the initial steps of ribosome biogenesis and other functions. Here, we review these biophysical insights in the context of the molecular and cell biology of the nucleolus. We discuss how nucleolar function is linked to its organization as a multiphase condensate and how dysregulation of this organization could provide insights into still poorly understood aspects of nucleolus-associated diseases, including cancer, ribosomopathies and neurodegeneration as well as ageing. We suggest that the LLPS model provides the starting point for a unifying quantitative framework for the assembly, structural maintenance and function of the nucleolus, with implications for gene regulation and ribonucleoprotein particle assembly throughout the nucleus. The LLPS concept is also likely useful in designing new therapeutic strategies to target nucleolar dysfunction. Protein trans-acting factors Among numerous microscopically visible nuclear sub- at the inner core where rRNA transcription occurs and Proteins important for structures, the nucleolus is the most prominent and proceeding towards the periphery (Fig.
    [Show full text]
  • Clusters of Bacterial RNA Polymerase Are Biomolecular Condensates That Assemble Through Liquid–Liquid Phase Separation
    Clusters of bacterial RNA polymerase are biomolecular condensates that assemble through liquid–liquid phase separation Anne-Marie Ladouceura,1, Baljyot Singh Parmarb,1, Stefan Biedzinskia, James Walla, S. Graydon Topea, David Cohna, Albright Kima, Nicolas Soubrya, Rodrigo Reyes-Lamothea, and Stephanie C. Webera,b,2 aDepartment of Biology, McGill University, Montreal, QC H3A 1B1, Canada; and bDepartment of Physics, McGill University, Montreal, QC H3A 2T8, Canada Edited by Richard A. Young, Massachusetts Institute of Technology, Cambridge, MA, and approved June 23, 2020 (received for review March 17, 2020) Once described as mere “bags of enzymes,” bacterial cells are in possibility that prokaryotes also use LLPS to compartmentalize fact highly organized, with many macromolecules exhibiting non- their cells. uniform localization patterns. Yet the physical and biochemical In eukaryotes, LLPS is mediated by proteins containing mul- mechanisms that govern this spatial heterogeneity remain largely tivalent domains and disordered regions (22–24) that bring unknown. Here, we identify liquid–liquid phase separation (LLPS) as molecules together into dynamic condensates through transient a mechanism for organizing clusters of RNA polymerase (RNAP) in interactions. Sequence analysis predicts that bacterial proteomes Escherichia coli . Using fluorescence imaging, we show that RNAP have a much lower frequency of disordered regions than quickly transitions from a dispersed to clustered localization pattern eukaryotic proteomes: Only 4.2% compared to 33%, respectively as cells enter log phase in nutrient-rich media. RNAP clusters are (25). This paucity of disorder raises doubts about the prevalence sensitive to hexanediol, a chemical that dissolves liquid-like com- of LLPS in this domain of life.
    [Show full text]
  • 2021.02.03.429226V1.Full.Pdf
    bioRxiv preprint doi: https://doi.org/10.1101/2021.02.03.429226; this version posted February 3, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. A modular platform for engineering function of natural and synthetic biomolecular condensates Keren Lasker1,*, Steven Boeynaems2,*, Vinson Lam3, Emma Stainton1, Maarten Jacquemyn4, Dirk Daelemans4, Elizabeth Villa3, Alex S. Holehouse5,6, Aaron D. Gitler2,#, Lucy Shapiro1,# 1Department of Developmental Biology, Stanford University School of Medicine, Stanford, CA 94305, USA 2Department of Genetics, Stanford University School of Medicine, Stanford, California 94305, USA 3Division of Biological Sciences, University of California San Diego, La Jolla, CA 92093, USA 4KU Leuven Department of Microbiology, Immunology, and Transplantation, Laboratory of Virology and Chemotherapy, Rega Institute, KU Leuven, 3000 Leuven, Belgium 5Department of Biochemistry and Molecular Biophysics, Washington University in St. Louis, St. Louis, MO 63110, USA. 6Center for Science and Engineering of Living Systems (CSELS), Washington University in St. Louis, St. Louis, MO 63130, USA *Equal contribution, #emails: [email protected], [email protected] Abstract Phase separation is emerging as a universal principle for how cells use dynamic subcompartmentalization to organize biochemical reactions in time and space1,2. Yet, whether the emergent physical properties of these biomolecular condensates are important
    [Show full text]
  • Phase Separation in Biology and Disease—
    Ann. N.Y. Acad. Sci. ISSN 0077-8923 ANNALS OF THE NEW YORK ACADEMY OF SCIENCES Special Issue: Annals Reports COMMENTARY Phase separation in biology and disease—a symposium report Jennifer Cable,1 Clifford Brangwynne,2 Geraldine Seydoux,3 David Cowburn,4 Rohit V. Pappu,5 Carlos A. Castañeda,6 Luke E. Berchowitz,7 Zhijuan Chen,8 Martin Jonikas,9 Abby Dernburg,10 Tanja Mittag,11 and Nicolas L. Fawzi12 1Science Writer, New York, New York. 2Department of Chemical and Biological Engineering, Princeton University, Princeton, New Jersey. 3Department of Molecular Biology and Genetics, Johns Hopkins University School of Medicine, Baltimore, Maryland. 4Departments of Biochemistry and Physiology and Biophysics, Albert Einstein College of Medicine, Bronx, New York. 5Department of Biomedical Engineering, Center for Science and Engineering of Living Systems, McKelvey School of Engineering, St. Louis, Missouri. 6Departments of Biology and Chemistry, Program in Neuroscience, Syracuse University, Syracuse, New York. 7Departments of Genetics and Development, Columbia University, New York, New York. 8University of Texas Southwestern Medical Center, Dallas, Texas. 9Department of Molecular Biology, Princeton University, Princeton, New Jersey. 10Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, California. 11Department of Structural Biology, St. Jude Children’s Research Hospital, Memphis, Tennessee. 12Department of Molecular Pharmacology, Physiology and Biotechnology, Brown University, Providence, Rhode Island Address for correspondence: [email protected] Phase separation of multivalent protein and RNA molecules enables cells the formation of reversible nonstoichio- metric, membraneless assemblies. These assemblies, referred to as biomolecular condensates, help with the spatial organization and compartmentalization of cellular matter. Each biomolecular condensate is defined by a distinct macromolecular composition.
    [Show full text]
  • Phase Separation in Biology
    Current Biology Magazine has not yet been applied to all Primer cytoplasm and the distribution of breeding programs, and there are these macromolecules is subject to still some major crop species (e.g., constant change. Early concepts to Phase separation soybean, tomato, sunfl ower) that are explain these density fl uctuations recalcitrant to the currently available in biology proposed a role for local synthesis, procedures, or for which present anchoring or active transport. Yet these protocols are not effi cient enough. Simon Alberti concepts are insuffi cient to explain The understanding of the maize in the complex organization of cells. situ system should help to extend Cells have to organize their complex We now realize that cells can only be the DH technology to more species biochemistry to regulate their properly understood if we also consider or breeding programs by either metabolism and respond to changes the collective properties of biological directly knocking out the functional in the environment. Traditionally, macromolecules. ortholog of the maize gene, or if intracellular organization has been Chemists have known for a long needed by transferring the cellular associated with compartments that time that macromolecules can have and molecular knowledge acquired are surrounded by lipid membranes. collective properties. The science of on maize. In addition to these However, in recent years, phase macromolecular collectives is called applications, the identifi cation of transitions have emerged as a novel colloidal chemistry. One example of a the NLD/MTL/ZmPLA1 gene offers form of cellular organization. Phase colloid is Jell-O, which is composed a unique opportunity to explore the transition is a physical process of colored water and gelatin, a large, many mysteries of double fertilization whereby a substance changes string-like protein.
    [Show full text]
  • New Perspectives on the Biogenesis of Viral Inclusion Bodies in Negative-Sense RNA Virus Infections
    cells Review New Perspectives on the Biogenesis of Viral Inclusion Bodies in Negative-Sense RNA Virus Infections Olga Dolnik , Gesche K. Gerresheim and Nadine Biedenkopf * Institute for Virology, Philipps-University Marburg, 35043 Marburg, Germany; [email protected] (O.D.); [email protected] (G.K.G.) * Correspondence: [email protected]; +49-(0)-64212825307 Abstract: Infections by negative strand RNA viruses (NSVs) induce the formation of viral inclusion bodies (IBs) in the host cell that segregate viral as well as cellular proteins to enable efficient viral replication. The induction of those membrane-less viral compartments leads inevitably to structural remodeling of the cellular architecture. Recent studies suggested that viral IBs have properties of biomolecular condensates (or liquid organelles), as have previously been shown for other membrane- less cellular compartments like stress granules or P-bodies. Biomolecular condensates are highly dynamic structures formed by liquid-liquid phase separation (LLPS). Key drivers for LLPS in cells are multivalent protein:protein and protein:RNA interactions leading to specialized areas in the cell that recruit molecules with similar properties, while other non-similar molecules are excluded. These typical features of cellular biomolecular condensates are also a common characteristic in the biogenesis of viral inclusion bodies. Viral IBs are predominantly induced by the expression of the viral nucleoprotein (N, NP) and phosphoprotein (P); both are characterized by a special protein architecture containing multiple disordered regions and RNA-binding domains that contribute to Citation: Dolnik, O.; Gerresheim, different protein functions. P keeps N soluble after expression to allow a concerted binding of N to G.K.; Biedenkopf, N.
    [Show full text]
  • RNA Impacts Formation of Biomolecular Condensates in the Nucleus
    Biomedical Research (Tokyo) 42 (4) 153–160, 2021 RNA impacts formation of biomolecular condensates in the nucleus Saho MATSUI and Ryu-Suke NOZAWA Division of Experimental Pathology, Cancer Institute of the Japanese Foundation for Cancer Research (JFCR), Ariake 3-8-31 Koto-ku, 135-8550 Tokyo, Japan (Received 10 May 2021; and accepted 11 June 2021) ABSTRACT Biomolecular condensates are membrane-less compartments that are formed through an assembly of proteins and nucleic acids in the cell. Dysregulation of biological condensates has been impli- cated in diseases such as neurodegeneration and cancer. Ribonucleic acid (RNA) is known to af- fect the assembly of proteins in vitro, if and how RNA is involved in regulating biomolecular condensates in cells is not well investigated. Here we examined two nuclear proteins, FUS and HP1α, in which RNA was found to have an opposite contribution for the assembly of these pro- teins. Reduction of nuclear RNA, by inhibiting the transcription, triggered assembly of FUS that had been distributed in the nucleoplasm, whereas it dispersed spontaneously formed HP1α assem- bly. Notably, the cell cycle-dependent phosphorylation-mimicking substitutions in HP1α promoted its assembly formation. These transcription inhibitor experiments are versatile to examine diverse roles of nuclear RNA in regulating biomolecular condensates, in both physiological and pathologi- cal conditions. uid phase composed of multiple soluble ingredients INTRODUCTION separating into different phases through their selec- Cells organize compartments in their interior to dif- tive and exclusive interactions (as a salad dressing ferentiate and facilitate biological processes. The nu- separates into oil- and water-phases). Through an cleus and mitochondria contain a lipid bilayer to analogous mechanism, selective assembly of bio- physically separate inside and outside and maintain molecules, such as proteins and nucleic acids, drives their internal biochemical environment.
    [Show full text]
  • Exploring Relationships Between the Density of Charged Tracts Within Disordered Regions and Phase Separation
    Pacific Symposium on Biocomputing 25:207-218(2020) Exploring Relationships between the Density of Charged Tracts within Disordered Regions and Phase Separation Ramiz Somjee Department of Structural Biology, St. Jude Children’s Research Hospital, 262 Danny Thomas Place Memphis, Tennessee 38105, USA; Department of Chemistry, Rhodes College, 2000 North Parkway Memphis, Tennessee 38112, USA; Email: [email protected] Diana M. Mitrea Department of Structural Biology, St. Jude Children’s Research Hospital, 262 Danny Thomas Place Memphis, Tennessee 38105, USA Email: [email protected] Richard W. Kriwacki† Department of Structural Biology, St. Jude Children’s Research Hospital, 262 Danny Thomas Place Memphis, Tennessee 38105, USA; Department of Microbiology, Immunology, and Biochemistry, University of Tennessee Health Sciences Center, 910 Madison Avenue, Memphis, Tennessee 38163, USA; Email: [email protected] Biomolecular condensates form through a process termed phase separation and play diverse roles throughout the cell. Proteins that undergo phase separation often have disordered regions that can engage in weak, multivalent interactions; however, our understanding of the sequence grammar that defines which proteins phase separate is far from complete. Here, we show that proteins that display a high density of charged tracts within intrinsically disordered regions are likely to be constituents of electrostatically organized biomolecular condensates. We scored the human proteome using an algorithm termed ABTdensity that quantifies the density of charged tracts and observed that proteins with more charged tracts are enriched in particular Gene Ontology annotations and, based upon analysis of interaction networks, cluster into distinct biomolecular condensates. These results suggest that electrostatically-driven, multivalent interactions involving charged tracts within disordered regions serve to organize certain biomolecular condensates through phase separation.
    [Show full text]
  • Programmable Viscoelasticity in Protein-RNA Condensates with Disordered Sticker- Spacer Polypeptides
    bioRxiv preprint doi: https://doi.org/10.1101/2021.01.24.427968; this version posted August 11, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. Programmable Viscoelasticity in Protein-RNA Condensates with Disordered Sticker- Spacer Polypeptides Ibraheem Alshareedah1, Mahdi Muhammad Moosa1, Matthew Pham2, Davit A. Potoyan2*, Priya R. Banerjee1* 1Department of Physics, University at Buffalo, Buffalo NY 14260, USA 2Department of Chemistry, Iowa State University, Ames IA 50011, USA *All correspondence could be addressed to: Priya R. Banerjee (email: [email protected]) Davit A. Potoyan (email: [email protected]) bioRxiv preprint doi: https://doi.org/10.1101/2021.01.24.427968; this version posted August 11, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. ABSTRACT Liquid-liquid phase separation of multivalent proteins and RNAs drives the formation of biomolecular condensates that facilitate membrane-free compartmentalization of subcellular processes. With recent advances, it is becoming increasingly clear that biomolecular condensates are network fluids with time-dependent material properties. Here, employing microrheology with optical tweezers, we reveal molecular determinants that govern the viscoelastic behavior of condensates formed by multivalent Arg/Gly-rich sticker-spacer polypeptides and RNA. These condensates behave as Maxwell fluids with an elastically-dominant rheological response at shorter timescales and a liquid-like behavior at longer timescales.
    [Show full text]