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

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.

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

10.1101/2020.05.31.122028 ◽

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.

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Versatile Regulator or Integrator RNA-binding Protein TLS/FUS Works for Phase Separation on Regulation of the Human Genome - In Relation to Liposarcoma and ALS

Biomedical Sciences ◽

10.11648/j.bs.20200601.12 ◽

2020 ◽

Vol 6 (1) ◽

pp. 5

Author(s):

Riki Kurokawa

Keyword(s):

Phase Separation ◽

Human Genome ◽

Rna Binding ◽

Binding Protein ◽

Rna Binding Protein

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

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.2114412118 ◽

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.

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

Journal of the American Chemical Society ◽

10.1021/jacs.1c04194 ◽

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

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

Nature Communications ◽

10.1038/s41467-021-21953-3 ◽

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.

Download Full-text