scholarly journals N-terminal sequences in matrin 3 mediate phase separation into droplet-like structures that recruit TDP43 variants lacking RNA binding elements

2019 ◽  
Vol 99 (7) ◽  
pp. 1030-1040 ◽  
Author(s):  
M. Carolina Gallego-Iradi ◽  
Haley Strunk ◽  
Anthony M. Crown ◽  
Rachel Davila ◽  
Hilda Brown ◽  
...  
Keyword(s):  
Phase Separation ◽  
Rna Binding ◽  
Matrin 3

scholarly journals The SARS-CoV-2 nucleocapsid phosphoprotein forms mutually exclusive condensates with RNA and the membrane-associated M protein

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Shan Lu ◽  
Qiaozhen Ye ◽  
Digvijay Singh ◽  
Yong Cao ◽  
Jolene K. Diedrich ◽  
...  
Keyword(s):  
Phase Separation ◽  
Potential Role ◽  
Rna Binding ◽  
Stress Granule ◽  
M Protein ◽  
Rich Region ◽  
Virion Assembly ◽  
N Protein ◽  
Intrinsically Disordered ◽  
Intrinsically Disordered Regions

AbstractThe multifunctional nucleocapsid (N) protein in SARS-CoV-2 binds the ~30 kb viral RNA genome to aid its packaging into the 80–90 nm membrane-enveloped virion. The N protein is composed of N-terminal RNA-binding and C-terminal dimerization domains that are flanked by three intrinsically disordered regions. Here we demonstrate that the N protein’s central disordered domain drives phase separation with RNA, and that phosphorylation of an adjacent serine/arginine rich region modulates the physical properties of the resulting condensates. In cells, N forms condensates that recruit the stress granule protein G3BP1, highlighting a potential role for N in G3BP1 sequestration and stress granule inhibition. The SARS-CoV-2 membrane (M) protein independently induces N protein phase separation, and three-component mixtures of N + M + RNA form condensates with mutually exclusive compartments containing N + M or N + RNA, including annular structures in which the M protein coats the outside of an N + RNA condensate. These findings support a model in which phase separation of the SARS-CoV-2 N protein contributes both to suppression of the G3BP1-dependent host immune response and to packaging genomic RNA during virion assembly.

Phase separation of RNA-binding protein promotes polymerase binding and transcription

2021 ◽  
Author(s):  
Wen Shao ◽  
Xianju Bi ◽  
Yixuan Pan ◽  
Boyang Gao ◽  
Jun Wu ◽  
...  
Keyword(s):  
Phase Separation ◽  
Rna Binding ◽  
Binding Protein ◽  
Rna Binding Protein ◽  
Polymerase Binding

scholarly journals TIA1 potentiates tau phase separation and promotes generation of toxic oligomeric tau

2021 ◽  
Vol 118 (9) ◽  
pp. e2014188118
Author(s):  
Peter E. A. Ash ◽  
Shuwen Lei ◽  
Jenifer Shattuck ◽  
Samantha Boudeau ◽  
Yari Carlomagno ◽  
...  
Keyword(s):  
Phase Separation ◽  
Polyethylene Glycol ◽  
Stress Response ◽  
Tau Protein ◽  
Rna Binding ◽  
Biological Activities ◽  
Rna Binding Protein ◽  
Physiological Conditions ◽  
Tau Oligomers

Tau protein plays an important role in the biology of stress granules and in the stress response of neurons, but the nature of these biochemical interactions is not known. Here we show that the interaction of tau with RNA and the RNA binding protein TIA1 is sufficient to drive phase separation of tau at physiological concentrations, without the requirement for artificial crowding agents such as polyethylene glycol (PEG). We further show that phase separation of tau in the presence of RNA and TIA1 generates abundant tau oligomers. Prior studies indicate that recombinant tau readily forms oligomers and fibrils in vitro in the presence of polyanionic agents, including RNA, but the resulting tau aggregates are not particularly toxic. We discover that tau oligomers generated during copartitioning with TIA1 are significantly more toxic than tau aggregates generated by incubation with RNA alone or phase-separated tau complexes generated by incubation with artificial crowding agents. This pathway identifies a potentially important source for generation of toxic tau oligomers in tau-related neurodegenerative diseases. Our results also reveal a general principle that phase-separated RBP droplets provide a vehicle for coassortment of selected proteins. Tau selectively copartitions with TIA1 under physiological conditions, emphasizing the importance of TIA1 for tau biology. Other RBPs, such as G3BP1, are able to copartition with tau, but this happens only in the presence of crowding agents. This type of selective mixing might provide a basis through which membraneless organelles bring together functionally relevant proteins to promote particular biological activities.

scholarly journals Double-stranded RNA drives SARS-CoV-2 nucleocapsid protein to undergo phase separation at specific temperatures

2021 ◽  
Author(s):  
Christine Roden ◽  
Yifan Dai ◽  
Ian Seim ◽  
Myungwoon Lee ◽  
Rachel Sealfon ◽  
...  
Keyword(s):  
Phase Separation ◽  
Rna Structure ◽  
Nucleocapsid Protein ◽  
Rna Binding ◽  
Genome Packaging ◽  
Rna Motifs ◽  
Double Stranded Rna ◽  
N Protein ◽  
Binding Domains

Betacoronavirus SARS-CoV-2 infections caused the global Covid-19 pandemic. The nucleocapsid protein (N-protein) is required for multiple steps in the betacoronavirus replication cycle. SARS-CoV-2-N-protein is known to undergo liquid-liquid phase separation (LLPS) with specific RNAs at particular temperatures to form condensates. We show that N-protein recognizes at least two separate and distinct RNA motifs, both of which require double-stranded RNA (dsRNA) for LLPS. These motifs are separately recognized by N-protein's two RNA binding domains (RBDs). Addition of dsRNA accelerates and modifies N-protein LLPS in vitro and in cells and controls the temperature condensates form. The abundance of dsRNA tunes N-protein-mediated translational repression and may confer a switch from translation to genome packaging. Thus, N-protein's two RBDs interact with separate dsRNA motifs, and these interactions impart distinct droplet properties that can support multiple viral functions. These experiments demonstrate a paradigm of how RNA structure can control the properties of biomolecular condensates.

scholarly journals RNA-binding and prion domains: the Yin and Yang of phase separation

2020 ◽  
Vol 48 (17) ◽  
pp. 9491-9504 ◽  
Author(s):  
Nieves Lorenzo Gotor ◽  
Alexandros Armaos ◽  
Giulia Calloni ◽  
Marc Torrent Burgas ◽  
R Martin Vabulas ◽  
...  
Keyword(s):  
Phase Separation ◽  
Computational Methods ◽  
Rna Binding ◽  
Stress Granule ◽  
Biochemical Reactions ◽  
Granule Protein ◽  
Binding Domains ◽  
Protein Partners ◽  
Yin And Yang ◽  
Opening Up

Abstract Proteins and RNAs assemble in membrane-less organelles that organize intracellular spaces and regulate biochemical reactions. The ability of proteins and RNAs to form condensates is encoded in their sequences, yet it is unknown which domains drive the phase separation (PS) process and what are their specific roles. Here, we systematically investigated the human and yeast proteomes to find regions promoting condensation. Using advanced computational methods to predict the PS propensity of proteins, we designed a set of experiments to investigate the contributions of Prion-Like Domains (PrLDs) and RNA-binding domains (RBDs). We found that one PrLD is sufficient to drive PS, whereas multiple RBDs are needed to modulate the dynamics of the assemblies. In the case of stress granule protein Pub1 we show that the PrLD promotes sequestration of protein partners and the RBD confers liquid-like behaviour to the condensate. Our work sheds light on the fine interplay between RBDs and PrLD to regulate formation of membrane-less organelles, opening up the avenue for their manipulation.

Abstract P4-05-09: Matrin 3: A novel micro-tubule associated RNA binding protein that acts as a potent tumor suppressor

2015 ◽  
Author(s):  
Panneerdoss Subbarayalu ◽  
Subapriya Rajamanickam ◽  
Suryavathi Viswanadhapalli ◽  
Nicholas Dybdal-Hargreaves ◽  
Santosh Timilsina ◽  
...  
Keyword(s):  
Tumor Suppressor ◽  
Rna Binding ◽  
Binding Protein ◽  
Rna Binding Protein ◽  
Matrin 3 ◽  
Potent Tumor

scholarly journals Zinc and Copper Ions Differentially Regulate Prion-Like Phase Separation Dynamics of Pan-Virus Nucleocapsid Biomolecular Condensates

Viruses ◽  
2020 ◽  
Vol 12 (10) ◽  
pp. 1179 ◽  
Author(s):  
Anne Monette ◽  
Andrew J. Mouland
Keyword(s):  
Phase Separation ◽  
Metal Ion ◽  
Rna Binding ◽  
Copper Ions ◽  
Virus Assembly ◽  
Neurological Diseases ◽  
Meta Analysis ◽  
Metal Homeostasis ◽  
Virus Infections ◽  
As Stress

Liquid-liquid phase separation (LLPS) is a rapidly growing research focus due to numerous demonstrations that many cellular proteins phase-separate to form biomolecular condensates (BMCs) that nucleate membraneless organelles (MLOs). A growing repertoire of mechanisms supporting BMC formation, composition, dynamics, and functions are becoming elucidated. BMCs are now appreciated as required for several steps of gene regulation, while their deregulation promotes pathological aggregates, such as stress granules (SGs) and insoluble irreversible plaques that are hallmarks of neurodegenerative diseases. Treatment of BMC-related diseases will greatly benefit from identification of therapeutics preventing pathological aggregates while sparing BMCs required for cellular functions. Numerous viruses that block SG assembly also utilize or engineer BMCs for their replication. While BMC formation first depends on prion-like disordered protein domains (PrLDs), metal ion-controlled RNA-binding domains (RBDs) also orchestrate their formation. Virus replication and viral genomic RNA (vRNA) packaging dynamics involving nucleocapsid (NC) proteins and their orthologs rely on Zinc (Zn) availability, while virus morphology and infectivity are negatively influenced by excess Copper (Cu). While virus infections modify physiological metal homeostasis towards an increased copper to zinc ratio (Cu/Zn), how and why they do this remains elusive. Following our recent finding that pan-retroviruses employ Zn for NC-mediated LLPS for virus assembly, we present a pan-virus bioinformatics and literature meta-analysis study identifying metal-based mechanisms linking virus-induced BMCs to neurodegenerative disease processes. We discover that conserved degree and placement of PrLDs juxtaposing metal-regulated RBDs are associated with disease-causing prion-like proteins and are common features of viral proteins responsible for virus capsid assembly and structure. Virus infections both modulate gene expression of metalloproteins and interfere with metal homeostasis, representing an additional virus strategy impeding physiological and cellular antiviral responses. Our analyses reveal that metal-coordinated virus NC protein PrLDs initiate LLPS that nucleate pan-virus assembly and contribute to their persistence as cell-free infectious aerosol droplets. Virus aerosol droplets and insoluble neurological disease aggregates should be eliminated by physiological or environmental metals that outcompete PrLD-bound metals. While environmental metals can control virus spreading via aerosol droplets, therapeutic interference with metals or metalloproteins represent additional attractive avenues against pan-virus infection and virus-exacerbated neurological diseases.

scholarly journals Nonclassical nuclear localization signals mediate nuclear import of CIRBP

2020 ◽  
Vol 117 (15) ◽  
pp. 8503-8514 ◽  
Author(s):  
Benjamin Bourgeois ◽  
Saskia Hutten ◽  
Benjamin Gottschalk ◽  
Mario Hofweber ◽  
Gesa Richter ◽  
...  
Keyword(s):  
Phase Separation ◽  
Nuclear Localization ◽  
Nuclear Import ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Specific Interaction ◽  
Stress Granule ◽  
Rich Region ◽  
Nuclear Localization Signals ◽  
Cargo Proteins

The specific interaction of importins with nuclear localization signals (NLSs) of cargo proteins not only mediates nuclear import but also, prevents their aberrant phase separation and stress granule recruitment in the cytoplasm. The importin Transportin-1 (TNPO1) plays a key role in the (patho-)physiology of both processes. Here, we report that both TNPO1 and Transportin-3 (TNPO3) recognize two nonclassical NLSs within the cold-inducible RNA-binding protein (CIRBP). Our biophysical investigations show that TNPO1 recognizes an arginine-glycine(-glycine) (RG/RGG)–rich region, whereas TNPO3 recognizes a region rich in arginine-serine-tyrosine (RSY) residues. These interactions regulate nuclear localization, phase separation, and stress granule recruitment of CIRBP in cells. The presence of both RG/RGG and RSY regions in numerous other RNA-binding proteins suggests that the interaction of TNPO1 and TNPO3 with these nonclassical NLSs may regulate the formation of membraneless organelles and subcellular localization of numerous proteins.

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.

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