Identification and molecular characterization of mutations in nucleocapsid phosphoprotein of SARS-CoV-2

SARS-CoV-2 genome encodes four structural proteins that include the spike glycoprotein, membrane protein, envelope protein and nucleocapsid phosphoprotein (N-protein). The N-protein interacts with viral genomic RNA and helps in packaging. As SARS-CoV-2 spread to almost all countries worldwide within 2–3 months, it also acquired mutations in its RNA genome. Therefore, this study was conducted with an aim to identify the variations present in N-protein of SARS-CoV-2. Here, we analysed 4,163 reported sequence of N-protein from United States of America (USA) and compared them with the first reported sequence from Wuhan, China. Our study identified 107 mutations that reside all over the N-protein. Further, we show the high rate of mutations in intrinsically disordered regions (IDRs) of N-protein. Our study show 45% residues of IDR2 harbour mutations. The RNA-binding domain (RBD) and dimerization domain of N-protein also have mutations at key residues. We further measured the effect of these mutations on N-protein stability and dynamicity and our data reveals that multiple mutations can cause considerable alterations. Altogether, our data strongly suggests that N-protein is one of the mutational hotspot proteins of SARS-CoV-2 that is changing rapidly and these mutations can potentially interferes with various aspects of N-protein functions including its interaction with RNA, oligomerization and signalling events.

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

10.1101/2020.07.30.228023 ◽

2020 ◽

Cited By ~ 1

Author(s):

Shan Lu ◽

Qiaozhen Ye ◽

Digvijay Singh ◽

Elizabeth Villa ◽

Don W. Cleveland ◽

...

Keyword(s):

Phase Separation ◽

Rna Binding ◽

M Protein ◽

Rna Packaging ◽

Dimerization Domain ◽

Virion Assembly ◽

N Protein ◽

Intrinsically Disordered ◽

Intrinsically Disordered Regions ◽

C Terminus

The 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 a centrally located 40 amino acid intrinsically disordered domain drives phase separation of N protein when bound to RNA, with the morphology of the resulting condensates affected by inclusion in the RNA of the putative SARS-CoV-2 packaging signal. The SARS-CoV-2 M protein, normally embedded in the virion membrane with its C-terminus extending into the virion core, independently induces N protein phase separation that is dependent on the N protein's C-terminal dimerization domain and disordered region. Three-component mixtures of N+M+RNA form condensates with mutually exclusive compartments containing N+M or N+RNA, including spherical 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 N protein with both the viral genomic RNA and the SARS-CoV-2 M protein facilitates RNA packaging and virion assembly.

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

Nature Communications ◽

10.1038/s41467-020-20768-y ◽

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.

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1H, 13C, and 15N backbone chemical shift assignments of the C-terminal dimerization domain of SARS-CoV-2 nucleocapsid protein

Biomolecular NMR Assignments ◽

10.1007/s12104-020-09995-y ◽

2020 ◽

Author(s):

Sophie M. Korn ◽

Roderick Lambertz ◽

Boris Fürtig ◽

Martin Hengesbach ◽

Frank Löhr ◽

...

Keyword(s):

Chemical Shift ◽

Nucleocapsid Protein ◽

Rna Binding ◽

Drug Binding ◽

Chemical Shift Assignments ◽

Binding Studies ◽

Dimerization Domain ◽

N Protein ◽

Intrinsically Disordered ◽

Backbone Chemical Shift

AbstractThe current outbreak of the highly infectious COVID-19 respiratory disease is caused by the novel coronavirus SARS-CoV-2 (Severe Acute Respiratory Syndrome Coronavirus 2). To fight the pandemic, the search for promising viral drug targets has become a cross-border common goal of the international biomedical research community. Within the international Covid19-NMR consortium, scientists support drug development against SARS-CoV-2 by providing publicly available NMR data on viral proteins and RNAs. The coronavirus nucleocapsid protein (N protein) is an RNA-binding protein involved in viral transcription and replication. Its primary function is the packaging of the viral RNA genome. The highly conserved architecture of the coronavirus N protein consists of an N-terminal RNA-binding domain (NTD), followed by an intrinsically disordered Serine/Arginine (SR)-rich linker and a C-terminal dimerization domain (CTD). Besides its involvement in oligomerization, the CTD of the N protein (N-CTD) is also able to bind to nucleic acids by itself, independent of the NTD. Here, we report the near-complete NMR backbone chemical shift assignments of the SARS-CoV-2 N-CTD to provide the basis for downstream applications, in particular site-resolved drug binding studies.

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Matrin3: Disorder and ALS Pathogenesis

Frontiers in Molecular Biosciences ◽

10.3389/fmolb.2021.794646 ◽

2022 ◽

Vol 8 ◽

Author(s):

Ahmed Salem ◽

Carter J. Wilson ◽

Benjamin S. Rutledge ◽

Allison Dilliott ◽

Sali Farhan ◽

...

Keyword(s):

Nuclear Export ◽

Binding Proteins ◽

Rna Binding ◽

Nuclear Dna ◽

Neurodegenerative Disorder ◽

Rna Binding Proteins ◽

Binding Protein ◽

Intrinsically Disordered ◽

Intrinsically Disordered Regions ◽

Disordered Regions

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disorder characterized by the degeneration of both upper and lower motor neurons in the brain and spinal cord. ALS is associated with protein misfolding and inclusion formation involving RNA-binding proteins, including TAR DNA-binding protein (TDP-43) and fused in sarcoma (FUS). The 125-kDa Matrin3 is a highly conserved nuclear DNA/RNA-binding protein that is implicated in many cellular processes, including binding and stabilizing mRNA, regulating mRNA nuclear export, modulating alternative splicing, and managing chromosomal distribution. Mutations in MATR3, the gene encoding Matrin3, have been identified as causal in familial ALS (fALS). Matrin3 lacks a prion-like domain that characterizes many other ALS-associated RNA-binding proteins, including TDP-43 and FUS, however, our bioinformatics analyses and preliminary studies document that Matrin3 contains long intrinsically disordered regions that may facilitate promiscuous interactions with many proteins and may contribute to its misfolding. In addition, these disordered regions in Matrin3 undergo numerous post-translational modifications, including phosphorylation, ubiquitination and acetylation that modulate the function and misfolding of the protein. Here we discuss the disordered nature of Matrin3 and review the factors that may promote its misfolding and aggregation, two elements that might explain its role in ALS pathogenesis.

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Interactions between the Intrinsically Disordered Regions of hnRNP-A2 and TDP-43 Accelerate TDP-43′s Conformational Transition

International Journal of Molecular Sciences ◽

10.3390/ijms21165930 ◽

2020 ◽

Vol 21 (16) ◽

pp. 5930

Author(s):

Wan-Chin Chiang ◽

Ming-Hsuan Lee ◽

Tsai-Chen Chen ◽

Jie-rong Huang

Keyword(s):

Intrinsically Disordered Proteins ◽

Rna Binding ◽

Conformational Transition ◽

Rna Binding Proteins ◽

Sequence Similarity ◽

Disordered Proteins ◽

General Mechanism ◽

Intrinsically Disordered ◽

Intrinsically Disordered Regions ◽

Disordered Regions

Most biological functions involve protein–protein interactions. Our understanding of these interactions is based mainly on those of structured proteins, because encounters between intrinsically disordered proteins (IDPs) or proteins with intrinsically disordered regions (IDRs) are much less studied, regardless of the fact that more than half eukaryotic proteins contain IDRs. RNA-binding proteins (RBPs) are a large family whose members almost all have IDRs in addition to RNA binding domains. These IDRs, having low sequence similarity, interact, but structural details on these interactions are still lacking. Here, using the IDRs of two RBPs (hnRNA-A2 and TDP-43) as a model, we demonstrate that the rate at which TDP-43′s IDR undergoes the neurodegenerative disease related α-helix-to-β-sheet transition increases in relation to the amount of hnRNP-A2′s IDR that is present. There are more than 1500 RBPs in human cells and most of them have IDRs. RBPs often join the same complexes to regulate genes. In addition to the structured RNA-recognition motifs, our study demonstrates a general mechanism through which RBPs may regulate each other’s functions through their IDRs.

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Distinct RNA polymerase transcripts direct the assembly of phase-separated DBC1 nuclear bodies in different cell lines

Molecular Biology of the Cell ◽

10.1091/mbc.e21-02-0081 ◽

2021 ◽

pp. mbc.E21-02-0081

Author(s):

Taro Mannen ◽

Masato Goto ◽

Takuya Yoshizawa ◽

Akio Yamashita ◽

Tetsuro Hirose ◽

...

Keyword(s):

Rna Polymerase ◽

Rna Binding ◽

Nuclear Body ◽

Nuclear Bodies ◽

Intrinsically Disordered ◽

Intrinsically Disordered Regions ◽

Binding Domains ◽

Additional Protein ◽

Protein Components

The mammalian cell nucleus is a highly organized organelle that contains membrane-less structures referred to as nuclear bodies (NBs). Some NBs carry specific RNA types that play architectural roles in their formation. Here, we show two types of RNase-sensitive DBC1-containing NBs: DBC1 nuclear body (DNB) in HCT116 cells and Sam68 nuclear body (SNB) in HeLa cells that exhibit phase-separated features and are constructed using RNA polymerase I or II transcripts in a cell type-specific manner. We identified additional protein components present in DNB by immunoprecipitation-mass spectrometry, some of which (DBC1 and HNRNPL) are required for DNB formation. The rescue experiment using the truncated HNRNPL mutants revealed that two RNA-binding domains and intrinsically disordered regions of HNRNPL play significant roles in DNB formation. All these domains of HNRNPL promote in vitro droplet formation, suggesting the need for multivalent interactions between HNRNPL and RNA as well as proteins in DNB formation.

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Energetic and structural features of SARS-CoV-2 N-protein co-assemblies with nucleic acids

10.1101/2021.02.08.430344 ◽

2021 ◽

Author(s):

Huaying Zhao ◽

Di Wu ◽

Ai Nguyen ◽

Yan Li ◽

Regina C. Adão ◽

...

Keyword(s):

Phase Separation ◽

Secondary Structure ◽

Protein Interactions ◽

Protein Secondary Structure ◽

Size Composition ◽

Structural Features ◽

N Protein ◽

Intrinsically Disordered ◽

Intrinsically Disordered Regions ◽

Ribonucleoprotein Particles

SummaryNucleocapsid (N) protein of the SARS-CoV-2 virus packages the viral genome into well-defined ribonucleoprotein particles, but the molecular pathway is still unclear. N-protein is dimeric and consists of two folded domains with nucleic acid (NA) binding sites, surrounded by intrinsically disordered regions that promote liquid-liquid phase separation. Here we use biophysical tools to study N-protein interactions with oligonucleotides of different length, examining the size, composition, secondary structure, and energetics of the resulting states. We observe formation of supramolecular clusters or nuclei preceding growth into phase-separated droplets. Short hexanucleotide NA forms compact 2:2 N-protein/NA complexes with reduced disorder. Longer oligonucleotides expose additional N-protein interactions and multi-valent protein-NA interactions, which generate higher-order mixed oligomers and simultaneously promote growth of droplets. Phase separation is accompanied by a significant increase in protein secondary structure, different from that caused by initial NA binding, which may contribute to the assembly of ribonucleoprotein particles within molecular condensates.

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Why do eukaryotic proteins contain more intrinsically disordered regions?

10.1101/270694 ◽

2018 ◽

Author(s):

Walter Basile ◽

Marco Salvatore ◽

Claudio Bassot ◽

Arne Elofsson

Keyword(s):

Amino Acids ◽

Amino Acid ◽

Intrinsic Disorder ◽

Intrinsically Disordered ◽

Intrinsically Disordered Regions ◽

Significant Difference ◽

Eukaryotic Proteins ◽

The Difference ◽

Almost All ◽

Disordered Regions

AbstractIntrinsic disorder is much more abundant in eukaryotic than in prokaryotic proteins. However, the reason behind this is unclear. It has been proposed that the disordered regions are functionally important for regulation in eukaryotes, but it has also been proposed that the difference is a result of lower selective pressure in eukaryotes. Almost all studies intrinsic disorder is predicted from the amino acid sequence of a protein. Therefore, there should exist an underlying difference in the amino acid distributions between eukaryotic and prokaryotic proteins causing the predicted difference in intrinsic disorder. To obtain a better understanding of why eukaryotic proteins contain more intrinsically disordered regions we compare proteins from complete eukaryotic and prokaryotic proteomes.Here, we show that the difference in intrinsic disorder origin from differences in the linker regions. Eukaryotic proteins have more extended linker regions and, in particular, the eukaryotic linker regions are more disordered. The average eukaryotic protein is about 500 residues long; it contains 250 residues in linker regions, of which 80 are disordered. In comparison, prokaryotic proteins are about 350 residues long and only have 100-110 residues in linker regions, and less than 10 of these are intrinsically disordered.Further, we show that there is no systematic increase in the frequency of disorder-promoting residues in eukaryotic linker regions. Instead, the difference in frequency of only three amino acids seems to lie behind the difference. The most significant difference is that eukaryotic linkers contain about 9% serine, while prokaryotic linkers have roughly 6.5%. Eukaryotic linkers also contain about 2% more proline and 2-3% fewer isoleucine residues. The reason why primarily these amino acids vary in frequency is not apparent, but it cannot be excluded that the difference is serine is related to the increased need for regulation through phosphorylation and that the proline difference is related to increase of eukaryotic specific repeats.

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Ebola Virus VP30 Is an RNA Binding Protein

Journal of Virology ◽

10.1128/jvi.02523-06 ◽

2007 ◽

Vol 81 (17) ◽

pp. 8967-8976 ◽

Cited By ~ 44

Author(s):

Sinu P. John ◽

Tan Wang ◽

Scott Steffen ◽

Sonia Longhi ◽

Connie S. Schmaljohn ◽

...

Keyword(s):

Binding Affinity ◽

Ebola Virus ◽

Rna Binding ◽

Transcriptional Start Site ◽

Direct Interaction ◽

Binding Activity ◽

Stem Loop ◽

Intrinsically Disordered ◽

Intrinsically Disordered Regions

ABSTRACT The Ebola virus (EBOV) genome encodes for several proteins that are necessary and sufficient for replication and transcription of the viral RNAs in vitro; NP, VP30, VP35, and L. VP30 acts in trans with an RNA secondary structure upstream of the first transcriptional start site to modulate transcription. Using a bioinformatics approach, we identified a region within the N terminus of VP30 with sequence features that typify intrinsically disordered regions and a putative RNA binding site. To experimentally assess the ability of VP30 to directly interact with the viral RNA, we purified recombinant EBOV VP30 to >90% homogeneity and assessed RNA binding by UV cross-linking and filter-binding assays. VP30 is a strongly acidophilic protein; RNA binding became stronger as pH was decreased. Zn2+, but not Mg2+, enhanced activity. Enhancement of transcription by VP30 requires a RNA stem-loop located within nucleotides 54 to 80 of the leader region. VP30 showed low binding affinity to the predicted stem-loop alone or to double-stranded RNA but showed a good binding affinity for the stem-loop when placed in the context of upstream and downstream sequences. To map the region responsible for interacting with RNA, we constructed, purified, and assayed a series of N-terminal deletion mutations of VP30 for RNA binding. The key amino acids supporting RNA binding activity map to residues 26 to 40, a region rich in arginine. Thus, we show for the first time the direct interaction of EBOV VP30 with RNA and the importance of the N-terminal region for binding RNA.

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A combined NMR and EPR investigation on the effect of the disordered RGG regions in the structure and the activity of the RRM domain of FUS

Scientific Reports ◽

10.1038/s41598-020-77899-x ◽

2020 ◽

Vol 10 (1) ◽

Author(s):

A. Bonucci ◽

M. G. Murrali ◽

L. Banci ◽

R. Pierattelli

Keyword(s):

Rna Binding ◽

Rna Binding Proteins ◽

Structural Disorder ◽

Rna Recognition Motif ◽

Stem Loop ◽

Intrinsically Disordered ◽

Intrinsically Disordered Regions ◽

Fused In Sarcoma ◽

Rrm Domain ◽

High Dynamics

AbstractStructural disorder represents a key feature in the mechanism of action of RNA-binding proteins (RBPs). Recent insights revealed that intrinsically disordered regions (IDRs) linking globular domains modulate their capability to interact with various sequences of RNA, but also regulate aggregation processes, stress-granules formation, and binding to other proteins. The FET protein family, which includes FUS (Fused in Sarcoma), EWG (Ewing Sarcoma) and TAF15 (TATA binding association factor 15) proteins, is a group of RBPs containing three different long IDRs characterized by the presence of RGG motifs. In this study, we present the characterization of a fragment of FUS comprising two RGG regions flanking the RNA Recognition Motif (RRM) alone and in the presence of a stem-loop RNA. From a combination of EPR and NMR spectroscopies, we established that the two RGG regions transiently interact with the RRM itself. These interactions may play a role in the recognition of stem-loop RNA, without a disorder-to-order transition but retaining high dynamics.

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