scholarly journals N‐terminal acetylation modestly enhances phase separation and reduces aggregation of the low‐complexity domain of RNA ‐binding protein FUS

2021 ◽  
Author(s):  
Anna S. Bock ◽  
Anastasia C. Murthy ◽  
Wai Shing Tang ◽  
Nina Jovic ◽  
Frank Shewmaker ◽  
...  
Keyword(s):  
Phase Separation ◽  
Rna Binding ◽  
Binding Protein ◽  
Rna Binding Protein ◽  
Low Complexity

scholarly journals The low-complexity domain of the FUS RNA binding protein self-assembles via the mutually exclusive use of two distinct cross-β cores

2021 ◽  
Vol 118 (42) ◽  
pp. e2114412118
Author(s):  
Masato Kato ◽  
Steven L. McKnight
Keyword(s):  
Phase Separation ◽  
Rna Binding ◽  
Binding Protein ◽  
Rna Binding Protein ◽  
Low Complexity ◽  
Terminal Region ◽  
Aqueous Environment ◽  
Fused In Sarcoma ◽  
Biochemical Assays ◽  
Biologic Function

The low-complexity (LC) domain of the fused in sarcoma (FUS) RNA binding protein self-associates in a manner causing phase separation from an aqueous environment. Incubation of the FUS LC domain under physiologically normal conditions of salt and pH leads to rapid formation of liquid-like droplets that mature into a gel-like state. Both examples of phase separation have enabled reductionist biochemical assays allowing discovery of an N-terminal region of 57 residues that assembles into a labile, cross-β structure. Here we provide evidence of a nonoverlapping, C-terminal region of the FUS LC domain that also forms specific cross-β interactions. We propose that biologic function of the FUS LC domain may operate via the mutually exclusive use of these N- and C-terminal cross-β cores. Neurodegenerative disease–causing mutations in the FUS LC domain are shown to imbalance the two cross-β cores, offering an unanticipated concept of LC domain function and dysfunction.

O-Linked-N-Acetylglucosaminylation of the RNA-Binding Protein EWS N-Terminal Low Complexity Region Reduces Phase Separation and Enhances Condensate Dynamics

2021 ◽  
Author(s):  
Michael L. Nosella ◽  
Maria Tereshchenko ◽  
Iva Pritišanac ◽  
P. Andrew Chong ◽  
Jeffrey A. Toretsky ◽  
...  
Keyword(s):  
Phase Separation ◽  
Rna Binding ◽  
Binding Protein ◽  
Rna Binding Protein ◽  
Low Complexity

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 The SARS-CoV-2 nucleocapsid protein is dynamic, disordered, and phase separates with RNA

2020 ◽  
Author(s):  
Jasmine Cubuk ◽  
Jhullian J. Alston ◽  
J. Jeremías Incicco ◽  
Sukrit Singh ◽  
Melissa D. Stuchell-Brereton ◽  
...  
Keyword(s):  
Phase Separation ◽  
Single Molecule ◽  
Protein Function ◽  
Rna Binding ◽  
Binding Protein ◽  
Rna Binding Protein ◽  
Genome Packaging ◽  
Binding Motifs ◽  
N Protein ◽  
Single Genome

AbstractThe SARS-CoV-2 nucleocapsid (N) protein is an abundant RNA binding protein critical for viral genome packaging, yet the molecular details that underlie this process are poorly understood. Here we combine single-molecule spectroscopy with all-atom simulations to uncover the molecular details that contribute to N protein function. N protein contains three dynamic disordered regions that house putative transiently-helical binding motifs. The two folded domains interact minimally such that full-length N protein is a flexible and multivalent RNA binding protein. N protein also undergoes liquid-liquid phase separation when mixed with RNA, and polymer theory predicts that the same multivalent interactions that drive phase separation also engender RNA compaction. We offer a simple symmetry-breaking model that provides a plausible route through which single-genome condensation preferentially occurs over phase separation, suggesting that phase separation offers a convenient macroscopic readout of a key nanoscopic interaction.

scholarly journals Fusion protein EWS-FLI1 is incorporated into a protein granule in cells

2020 ◽  
Author(s):  
Nasiha S. Ahmed ◽  
Lucas M. Harrell ◽  
Jacob C. Schwartz
Keyword(s):  
Phase Separation ◽  
Fusion Protein ◽  
Ewing Sarcoma ◽  
Rna Binding ◽  
Binding Protein ◽  
Rna Binding Protein ◽  
Rna Pol Ii ◽  
Pol Ii ◽  
Protein Granules ◽  
In Cells

ABSTRACTEwing sarcoma is driven by fusion proteins containing a low complexity (LC) domain that is intrinsically disordered and a powerful transcriptional regulator. The most common fusion protein found in Ewing sarcoma, EWS-FLI1, takes its LC domain from the RNA-binding protein EWSR1 (Ewing Sarcoma RNA-binding protein 1) and a DNA-binding domain from the transcription factor FLI1 (Friend Leukemia Virus Integration 1). EWS-FLI1 binds RNA polymerase II (RNA Pol II) and can self-assemble through a process known as phase separation. The ability of self-oligomerizing RNA-binding proteins like EWSR1 to assemble into ribonucleoprotein granules in cells has received significant attention but the role of phase separation in EWS-FLI1 activity is less understood. We investigated the intersecting roles of EWSR1 and EWS-FLI1 to control gene expression and tumorigenic cell growth in Ewing sarcoma. We also studied interactions among EWS-FLI1, EWSR1, and RNA Pol II. We applied a crosslinking approach to demonstrate the incorporation of EWSR1 and RNA Pol II into protein granules in cells. We also identified protein granules in cells associated with the fusion protein, EWS-FLI1. Interactions through the LC domain, which allow EWS-FLI1 to bind EWSR1 and RNA Pol II, were found to be required for inclusion into the cellular granules observed by TEM. The physical characterization of EWS-FLI1 assemblies reported here offers insight into a large protein assembly that may allow EWS-FLI1 to engage its wide network of protein partners while driving tumorigenesis.

scholarly journals The SARS-CoV-2 nucleocapsid protein is dynamic, disordered, and phase separates with RNA

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Jasmine Cubuk ◽  
Jhullian J. Alston ◽  
J. Jeremías Incicco ◽  
Sukrit Singh ◽  
Melissa D. Stuchell-Brereton ◽  
...  
Keyword(s):  
Phase Separation ◽  
Single Molecule ◽  
Protein Function ◽  
Rna Binding ◽  
Binding Protein ◽  
Rna Binding Protein ◽  
Genome Packaging ◽  
Binding Motifs ◽  
N Protein ◽  
Single Genome

AbstractThe SARS-CoV-2 nucleocapsid (N) protein is an abundant RNA-binding protein critical for viral genome packaging, yet the molecular details that underlie this process are poorly understood. Here we combine single-molecule spectroscopy with all-atom simulations to uncover the molecular details that contribute to N protein function. N protein contains three dynamic disordered regions that house putative transiently-helical binding motifs. The two folded domains interact minimally such that full-length N protein is a flexible and multivalent RNA-binding protein. N protein also undergoes liquid-liquid phase separation when mixed with RNA, and polymer theory predicts that the same multivalent interactions that drive phase separation also engender RNA compaction. We offer a simple symmetry-breaking model that provides a plausible route through which single-genome condensation preferentially occurs over phase separation, suggesting that phase separation offers a convenient macroscopic readout of a key nanoscopic interaction.

scholarly journals Regulation of zebrafish dorsoventral patterning by phase separation of RNA-binding protein Rbm14

2019 ◽  
Vol 5 (1) ◽  
Author(s):  
Yue Xiao ◽  
Jiehui Chen ◽  
Yihan Wan ◽  
Qi Gao ◽  
Naihe Jing ◽  
...  
Keyword(s):  
Phase Separation ◽  
Rna Binding ◽  
Binding Protein ◽  
Rna Binding Protein ◽  
Dorsoventral Patterning

scholarly journals Low-complexity domain of U1-70K modulates phase separation and aggregation through distinctive basic-acidic motifs

2019 ◽  
Vol 5 (11) ◽  
pp. eaax5349 ◽  
Author(s):  
Song Xue ◽  
Rui Gong ◽  
Fanqi He ◽  
Yanqin Li ◽  
Yunjia Wang ◽  
...  
Keyword(s):  
Phase Separation ◽  
Liquid Phase ◽  
Rna Binding ◽  
Rna Binding Protein ◽  
Low Complexity ◽  
Protein Domain ◽  
Shed Light ◽  
Liquid Liquid Phase Separation

Liquid-liquid phase separation (LLPS) facilitates the formation of functional membraneless organelles and recent reports have linked this phenomenon to protein aggregation in neurodegenerative diseases. Understanding the mechanism of LLPS and its regulation thus promises to shed light on the pathogenesis of these conditions. The RNA-binding protein U1-70K, which aggregates in brains of Alzheimer’s disease patients, is considered a potential target for Alzheimer’s therapy. Here, we report that two fragments in the low-complexity (LC) domain of U1-70K can undergo LLPS. We have demonstrated that the repetitive basic-acidic motifs in these fragments induce nucleotide-independent phase separation and initiate aggregation in vitro. We also have confirmed that LLPS and aggregation occur in vivo and that the content of ampholytic motifs in a protein domain determines the transition between droplets and aggregation, providing insights into the mechanism underlying the formation of diverse assembly states.

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