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 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 FUS-dependent liquid–liquid phase separation is important for DNA repair initiation

2021 ◽  
Vol 220 (5) ◽  
Author(s):  
Brunno R. Levone ◽  
Silvia C. Lenzken ◽  
Marco Antonaci ◽  
Andreas Maiser ◽  
Alexander Rapp ◽  
...  
Keyword(s):  
Dna Damage ◽  
Dna Repair ◽  
Phase Separation ◽  
Liquid Phase ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Liquid Phase Separation ◽  
Structured Illumination Microscopy ◽  
Liquid Liquid Phase Separation

RNA-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, and 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 superresolution structured illumination microscopy, we demonstrate that the absence of FUS impairs the proper arrangement of γH2AX nanofoci 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 DSB repair complexes.

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 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 Nucleocapsid protein of SARS-CoV-2 phase separates into RNA-rich polymerase-containing condensates

2020 ◽  
Author(s):  
Adriana Savastano ◽  
Alain Ibáñez de Opakua ◽  
Marija Rankovic ◽  
Markus Zweckstetter
Keyword(s):  
Phase Separation ◽  
Liquid Phase ◽  
Nucleocapsid Protein ◽  
Etiologic Agent ◽  
Liquid Phase Separation ◽  
Viral Rna ◽  
Cytoplasmic Rna ◽  
Infected Cells ◽  
Liquid Liquid Phase Separation

SUMMARYThe etiologic agent of the Covid-19 pandemic is the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The viral membrane of SARS-CoV-2 surrounds a helical nucleocapsid in which the viral genome is encapsulated by the nucleocapsid protein. The nucleocapsid protein of SARS-CoV-2 is produced at high levels within infected cells, enhances the efficiency of viral RNA transcription and is essential for viral replication. Here we show that RNA induces cooperative liquid-liquid phase separation of the SARS-CoV-2 nucleocapsid protein. In agreement with its ability to phase separate in vitro, we show that the protein associates in cells with stress granules, cytoplasmic RNA/protein granules that form through liquid-liquid phase separation and are modulated by viruses to maximize replication efficiency. Liquid-liquid phase separation generates high-density protein/RNA condensates that recruit the RNA-dependent RNA polymerase complex of SARS-CoV-2 providing a mechanism for efficient transcription of viral RNA. Inhibition of RNA-induced phase separation of the nucleocapsid protein by small molecules or biologics thus can interfere with a key step in the SARS-CoV-2 replication cycle.

scholarly journals Nucleocapsid protein of SARS-CoV-2 phase separates into RNA-rich polymerase-containing condensates

2020 ◽  
Vol 11 (1) ◽  
Author(s):  
Adriana Savastano ◽  
Alain Ibáñez de Opakua ◽  
Marija Rankovic ◽  
Markus Zweckstetter
Keyword(s):  
Phase Separation ◽  
Liquid Phase ◽  
Nucleocapsid Protein ◽  
Etiologic Agent ◽  
Liquid Phase Separation ◽  
Viral Rna ◽  
Cytoplasmic Rna ◽  
Infected Cells ◽  
Liquid Liquid Phase Separation

AbstractThe etiologic agent of the Covid-19 pandemic is the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The viral membrane of SARS-CoV-2 surrounds a helical nucleocapsid in which the viral genome is encapsulated by the nucleocapsid protein. The nucleocapsid protein of SARS-CoV-2 is produced at high levels within infected cells, enhances the efficiency of viral RNA transcription, and is essential for viral replication. Here, we show that RNA induces cooperative liquid–liquid phase separation of the SARS-CoV-2 nucleocapsid protein. In agreement with its ability to phase separate in vitro, we show that the protein associates in cells with stress granules, cytoplasmic RNA/protein granules that form through liquid-liquid phase separation and are modulated by viruses to maximize replication efficiency. Liquid–liquid phase separation generates high-density protein/RNA condensates that recruit the RNA-dependent RNA polymerase complex of SARS-CoV-2 providing a mechanism for efficient transcription of viral RNA. Inhibition of RNA-induced phase separation of the nucleocapsid protein by small molecules or biologics thus can interfere with a key step in the SARS-CoV-2 replication cycle.

scholarly journals Targeting NSD2-mediated SRC-3 liquid–liquid phase separation sensitizes bortezomib treatment in multiple myeloma

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Jing Liu ◽  
Ying Xie ◽  
Jing Guo ◽  
Xin Li ◽  
Jingjing Wang ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Drug Resistance ◽  
Phase Separation ◽  
Liquid Phase ◽  
Liquid Phase Separation ◽  
Acquired Drug Resistance ◽  
Bortezomib Treatment ◽  
Liquid Liquid Phase Separation

AbstractDevelopment of chemoresistance is the main reason for failure of clinical management of multiple myeloma (MM), but the genetic and epigenetic aberrations that interact to confer such chemoresistance remains unknown. In the present study, we find that high steroid receptor coactivator-3 (SRC-3) expression is correlated with relapse/refractory and poor outcomes in MM patients treated with bortezomib (BTZ)-based regimens. Furthermore, in immortalized cell lines, high SRC-3 enhances resistance to proteasome inhibitor (PI)-induced apoptosis. Overexpressed histone methyltransferase NSD2 in patients bearing a t(4;14) translocation or in BTZ-resistant MM cells coordinates elevated SRC-3 by enhancing its liquid–liquid phase separation to supranormally modify histone H3 lysine 36 dimethylation (H3K36me2) modifications on promoters of anti-apoptotic genes. Targeting SRC-3 or interference of its interactions with NSD2 using a newly developed inhibitor, SI-2, sensitizes BTZ treatment and overcomes drug resistance both in vitro and in vivo. Taken together, our findings elucidate a previously unrecognized orchestration of SRC-3 and NSD2 in acquired drug resistance of MM and suggest that SI-2 may be efficacious for overcoming drug resistance in MM patients.

scholarly journals G-quadruplex DNA inhibits unwinding activity but promotes liquid–liquid phase separation by the DEAD-box helicase Ded1p

2021 ◽  
Author(s):  
Jun Gao ◽  
Zhaofeng Gao ◽  
Andrea A. Putnam ◽  
Alicia K. Byrd ◽  
Sarah L. Venus ◽  
...  
Keyword(s):  
Phase Separation ◽  
Liquid Phase ◽  
Liquid Phase Separation ◽  
Intrinsically Disordered ◽  
Dead Box ◽  
G4 Dna ◽  
G Quadruplex ◽  
The Dead ◽  
Liquid Liquid Phase Separation

G-quadruplex (G4) DNA inhibits RNA unwinding activity but promotes liquid–liquid phase separation of the DEAD-box helicase Ded1p in vitro and in cells. This highlights multifaceted effects of G4DNA on an enzyme with intrinsically disordered domains.

scholarly journals Liquid-liquid phase separation of the chromosomal passenger complex drives parallel bundling of midzone microtubules

2022 ◽  
Author(s):  
Ewa Niedzialkowska ◽  
Tan M Truong ◽  
Luke A Eldredge ◽  
Stefanie Redemann ◽  
Denis Chretien ◽  
...  
Keyword(s):  
Phase Separation ◽  
Liquid Phase ◽  
Chromosome Segregation ◽  
Dynamic Structure ◽  
Liquid Phase Separation ◽  
Chromosomal Passenger Complex ◽  
Chromosomal Passenger ◽  
Spindle Midzone ◽  
Liquid Liquid Phase Separation

The spindle midzone is a dynamic structure that forms during anaphase, mediates chromosome segregation, and provides a signaling platform to position the cleavage furrow. The spindle midzone comprises two antiparallel bundles of microtubules (MTs) but the process of their formation is poorly understood. Here, we show that the Chromosomal Passenger Complex (CPC) undergoes liquid-liquid phase separation (LLPS) to generate parallel MT bundles in vitro when incubated with free tubulin and GTP. MT bundles emerge from CPC droplets with protruding minus-ends that then grow into long, tapered MT structures. During this growth, the CPC in condensates apparently reorganize to coat and bundle the resulting MT structures. CPC mutants attenuated for LLPS or MT binding prevented the generation of parallel MT bundles in vitro and reduced the number of MTs present at spindle midzones in HeLa cells. Our data uncovers a kinase-independent function of the CPC and provides models for how cells generate parallel-bundled MT structures that are important for the assembly of the mitotic spindle.

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