scholarly journals Tandem RNA binding sites induce self-association of the stress granule marker protein TIA-1

2021 ◽  
Author(s):  
Fionna E Loughlin ◽  
Danella L West ◽  
Menachem J Gunzburg ◽  
Saboora Waris ◽  
Simon A Crawford ◽  
...  
Keyword(s):  
Phase Separation ◽  
Nucleic Acid ◽  
Liquid Phase ◽  
Binding Sites ◽  
Rna Binding ◽  
Liquid Phase Separation ◽  
Stress Granule ◽  
Granule Formation ◽  
Self Association ◽  
Liquid Liquid Phase Separation

Abstract TIA-1 is an RNA-binding protein that sequesters target RNA into stress granules under conditions of cellular stress. Promotion of stress granule formation by TIA-1 depends upon self-association of its prion-like domain that facilitates liquid-liquid phase separation and is thought to be enhanced via RNA binding. However, the mechanisms underlying the influence of RNA on TIA-1 self-association have not been previously demonstrated. Here we have investigated the self-associating properties of full-length TIA-1 in the presence of designed and native TIA-1 nucleic acid binding sites in vitro, monitoring phase separation, fibril formation and shape. We show that single stranded RNA and DNA induce liquid-liquid phase separation of TIA-1 in a multisite, sequence-specific manner and also efficiently promote formation of amyloid-like fibrils. Although RNA binding to a single site induces a small conformational change in TIA-1, this alone does not enhance phase separation of TIA-1. Tandem binding sites are required to enhance phase separation of TIA-1 and this is finely tuned by the protein:binding site stoichiometry rather than nucleic acid length. Native tandem TIA-1 binding sites within the 3′ UTR of p53 mRNA also efficiently enhance phase separation of TIA-1 and thus may potentially act as potent nucleation sites for stress granule assembly.

scholarly journals Polymer Stiffness Regulates Multivalent Binding and Liquid-Liquid Phase Separation

2020 ◽  
Author(s):  
E. Zumbro ◽  
A. Alexander-Katz
Keyword(s):  
Phase Separation ◽  
Liquid Phase ◽  
Rational Design ◽  
Rna Binding ◽  
Liquid Phase Separation ◽  
Random Coil ◽  
Small Target ◽  
Toxic Protein ◽  
Multivalent Binding ◽  
Liquid Liquid Phase Separation

AbstractMultivalent binding is essential to many biological processes because it builds high affinity bonds by using several weak binding interactions simultaneously. Multivalent polymers have shown promise as inhibitors of toxins and other pathogens, and they are important components in the formation of biocondensates. Explaining how structural features of these polymers change their binding and subsequent control of phase separation is critical to designing better pathogen inhibitors and also to understanding diseases associated with membraneless organelles. In this work, we will examine the binding of a multivalent polymer to a small target. This scenario could represent a polymeric inhibitor binding to a toxic protein or RNA binding to an RNA-binding protein in the case of liquid-liquid phase separation. We use simulation and theory to show that flexible random-coil polymers bind more strongly than stiff rod-like polymers and that flexible polymers nucleate condensed phases at lower energies than their rigid analogues. We hope these results will provide insight into the rational design of polymeric inhibitors and improve understanding of membraneless organelles.Statement of SignificanceMultivalent polymers are essential for many biological systems, including targeting pathogens and controlling the formation of liquid-liquid phase separated biocondensates. Here, we explain how increasing polymer stiffness can reduce multivalent binding affinity to a small target such as a toxic protein and how modulating polymer stiffness can change the phase boundary for liquid-liquid phase separation. These results have implications for designing stronger pathogen inhibitors and provide insights on neurodegenerative diseases associated with abnormal biocondensate formation.

scholarly journals Liquid-Liquid Phase Separation of Patchy Particles Illuminates Diverse Effects of Regulatory Components on Protein Droplet Formation

2018 ◽  
Author(s):  
Valery Nguemaha ◽  
Huan-Xiang Zhou
Keyword(s):  
Phase Separation ◽  
Liquid Phase ◽  
Rna Binding ◽  
Liquid Phase Separation ◽  
Steric Repulsion ◽  
Cellular Functions ◽  
Binding Domains ◽  
Patchy Particles ◽  
Droplet Phase ◽  
Liquid Liquid Phase Separation

AbstractRecently many cellular functions have been associated with membraneless organelles, or protein droplets, formed by liquid-liquid phase separation (LLPS). Proteins in these droplets often contain RNA-binding domains, but the effects of RNA on LLPS have been controversial. To gain better understanding on the roles of RNA, here we used Gibbs-ensemble simulations to determine phase diagrams of two-component patchy particles, as models for mixtures of proteins with RNA or other regulatory components. Protein-like particles have four patches, with attraction strength εPP; regulatory particles experience mutual steric repulsion but have two attractive patches toward proteins, with the strength εPR tunable. At low εPR, the regulator, due to steric repulsion, preferentially partitions in the dispersed phase, thereby displacing the protein into the droplet phase and promoting LLPS. At moderate εPR, the regulator starts to partition and displace the protein in the droplet phase, but only to weaken bonding networks and thereby suppress LLPS. At εPR > εPP, the enhanced bonding ability of the regulator initially promotes LLPS, but at higher amounts, the resulting displacement of the protein suppresses LLPS. These results illustrate how RNA can have disparate effects on LLPS, thus able to perform diverse functions in different organelles.

scholarly journals SARS-CoV-2 nucleocapsid protein undergoes liquid-liquid phase separation stimulated by RNA and partitions into phases of human ribonucleoproteins

2020 ◽  
Author(s):  
Theodora Myrto Perdikari ◽  
Anastasia C. Murthy ◽  
Veronica H. Ryan ◽  
Scott Watters ◽  
Mandar T. Naik ◽  
...  
Keyword(s):  
Phase Separation ◽  
Liquid Phase ◽  
Nucleocapsid Protein ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Liquid Phase Separation ◽  
Host Protein ◽  
Host Cells ◽  
Liquid Liquid Phase Separation

AbstractTightly packed complexes of nucleocapsid protein and genomic RNA form the core of viruses and may assemble within viral factories, dynamic compartments formed within the host cells. Here, we examine the possibility that the multivalent RNA-binding nucleocapsid protein (N) from the severe acute respiratory syndrome coronavirus (SARS-CoV-2) compacts RNA via protein-RNA liquid-liquid phase separation (LLPS) and that N interactions with host RNA-binding proteins are mediated by phase separation. To this end, we created a construct expressing recombinant N fused to a N-terminal maltose binding protein tag which helps keep the oligomeric N soluble for purification. Using in vitro phase separation assays, we find that N is assembly-prone and phase separates avidly. Phase separation is modulated by addition of RNA and changes in pH and is disfavored at high concentrations of salt. Furthermore, N enters into in vitro phase separated condensates of full-length human hnRNPs (TDP-43, FUS, and hnRNPA2) and their low complexity domains (LCs). However, N partitioning into the LC of FUS, but not TDP-43 or hnRNPA2, requires cleavage of the solubilizing MBP fusion. Hence, LLPS may be an essential mechanism used for SARS-CoV-2 and other RNA viral genome packing and host protein co-opting, functions necessary for viral replication and hence infectivity.

scholarly journals TDP-43 α-helical structure tunes liquid–liquid phase separation and function

2020 ◽  
Vol 117 (11) ◽  
pp. 5883-5894 ◽  
Author(s):  
Alexander E. Conicella ◽  
Gregory L. Dignon ◽  
Gül H. Zerze ◽  
Hermann Broder Schmidt ◽  
Alexandra M. D’Ordine ◽  
...  
Keyword(s):  
Phase Separation ◽  
Liquid Phase ◽  
Structural Changes ◽  
Rna Binding ◽  
Helical Structure ◽  
Liquid Phase Separation ◽  
Control Assembly ◽  
And Function ◽  
Liquid Liquid Phase Separation

Liquid–liquid phase separation (LLPS) is involved in the formation of membraneless organelles (MLOs) associated with RNA processing. The RNA-binding protein TDP-43 is present in several MLOs, undergoes LLPS, and has been linked to the pathogenesis of amyotrophic lateral sclerosis (ALS). While some ALS-associated mutations in TDP-43 disrupt self-interaction and function, here we show that designed single mutations can enhance TDP-43 assembly and function via modulating helical structure. Using molecular simulation and NMR spectroscopy, we observe large structural changes upon dimerization of TDP-43. Two conserved glycine residues (G335 and G338) are potent inhibitors of helical extension and helix–helix interaction, which are removed in part by variants at these positions, including the ALS-associated G335D. Substitution to helix-enhancing alanine at either of these positions dramatically enhances phase separation in vitro and decreases fluidity of phase-separated TDP-43 reporter compartments in cells. Furthermore, G335A increases TDP-43 splicing function in a minigene assay. Therefore, the TDP-43 helical region serves as a short but uniquely tunable module where application of biophysical principles can precisely control assembly and function in cellular and synthetic biology applications of LLPS.

scholarly journals FUS-dependent liquid-liquid phase separation is an early event in double-strand break repair

2019 ◽  
Author(s):  
Brunno R. Levone ◽  
Silvia C. Lenzken ◽  
Marco Antonaci ◽  
Andreas Maiser ◽  
Alexander Rapp ◽  
...  
Keyword(s):  
Dna Damage ◽  
Phase Separation ◽  
Liquid Phase ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Liquid Phase Separation ◽  
Early Event ◽  
Structured Illumination Microscopy ◽  
Liquid Liquid Phase Separation

AbstractRNA-binding proteins (RBPs) are emerging as important effectors of the cellular DNA damage response (DDR). The RBP FUS is implicated in RNA metabolism and DNA repair, and it undergoes reversible liquid-liquid phase separation (LLPS) in vitro. Here, we demonstrate that FUS-dependent LLPS is necessary for the initiation of the DDR. Using laser microirradiation in FUS-knockout cells, we show that FUS is required for the recruitment to DNA damage sites of the DDR factors KU80, NBS1, 53BP1, and of SFPQ, another RBP implicated in the DDR. The relocation of KU80, NBS1, and SFPQ is similarly impaired by LLPS inhibitors, or LLPS-deficient FUS variants. We also show that LLPS is necessary for efficient γH2AX foci formation. Finally, using super-resolution structured illumination microscopy, we demonstrate that the absence of FUS impairs the proper arrangement of γH2AX nano-foci into higher-order clusters. These findings demonstrate the early requirement for FUS-dependent LLPS in the activation of the DDR and the proper assembly of DSBs repair complexes.

scholarly journals Phase-specific RNA accumulation and duplex thermodynamics in multiphase coacervate models for membraneless organelles

2021 ◽  
Author(s):  
Saehyun Choi ◽  
Philip C. Bevilacqua ◽  
Christine D. Keating
Keyword(s):  
Phase Separation ◽  
Liquid Phase ◽  
Binding Sites ◽  
Rna Binding ◽  
Multiphase Model ◽  
Duplex Stability ◽  
Functional Consequences ◽  
Important Means ◽  
Rna Accumulation ◽  
Liquid Liquid Phase Separation

Liquid-liquid phase separation has emerged as an important means of intracellular RNA compartmentalization. Some membraneless organelles host two or more compartments serving different putative biochemical roles; the mechanisms for, and functional consequences of, this subcompartmentalization are not yet well understood. Here, we show that adjacent phases of decapeptide-based multiphase model membraneless organelles differ markedly in their interactions with RNA. Additionally, their coexistence introduces new equilibria that alter RNA duplex stability and RNA sorting by hybridization state. These effects require neither biospecific RNA binding sites nor full-length proteins. As such, they are general and point to more primitive versions of mechanisms operating in extant biology that could aid understanding and enable design of functional artificial membraneless organelles.

scholarly journals Microtubules as platforms for probing liquid–liquid phase separation in cells – application to RNA-binding proteins

2018 ◽  
Vol 131 (11) ◽  
pp. jcs214692 ◽  
Author(s):  
Alexandre Maucuer ◽  
Bénédicte Desforges ◽  
Vandana Joshi ◽  
Mirela Boca ◽  
Dmitry A. Kretov ◽  
...  
Keyword(s):  
Phase Separation ◽  
Liquid Phase ◽  
Binding Proteins ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Liquid Phase Separation ◽  
Liquid Liquid Phase Separation ◽  
In Cells

scholarly journals CRISPR-based DNA and RNA detection with liquid-liquid phase separation

2018 ◽  
Author(s):  
Willem Kasper Spoelstra ◽  
Jeroen M. Jacques ◽  
Franklin L. Nobrega ◽  
Anna C. Haagsma ◽  
Marileen Dogterom ◽  
...  
Keyword(s):  
Phase Separation ◽  
Nucleic Acid ◽  
Liquid Phase ◽  
Clinical Diagnostics ◽  
Liquid Phase Separation ◽  
Molecular Diagnostic ◽  
Target Sequence ◽  
Dna And Rna ◽  
Wide Range ◽  
Liquid Liquid Phase Separation

AbstractThe ability to detect specific nucleic acid sequences allows for a wide range of applications including identification of pathogens, clinical diagnostics, and genotyping. CRISPR-Cas proteins Cas12a and Cas13a are RNA-guided endonucleases that bind and cleave specific DNA and RNA sequences, respectively. After recognition of a target sequence both enzymes activate a unique, indiscriminate nucleic acid cleavage activity, which has been exploited for detection of sequence specific nucleotides using labelled reporter molecules. We here present a label-free detection approach that uses a readout based on solution turbidity caused by liquid-liquid phase separation (LLPS). Turbidity arises from coacervates of positively charged polyelectrolytes with long poly(dT) or poly(U) oligonucleotides. In the presence of a target sequence, long oligonucleotides are progressively shortened, changing the solution from turbid to transparent. We explain how oligonucleotide cleavage resolves LLPS by using a mathematical model which we validate with poly(dT) phase separation experiments. The deployment of LLPS complements CRISPR-based molecular diagnostic applications and facilitates easy and low-cost nucleotide sequence detection.

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