Protein-RNA condensation kinetics via filamentous nanoclusters DOI Creative Commons

Ramón Peralta Martínez,

Araceli Visentín, Mariano Salgueiro

и другие.

bioRxiv (Cold Spring Harbor Laboratory), Год журнала: 2024, Номер unknown

Опубликована: Ноя. 8, 2024

Abstract Protein-RNA phase separation is at the center of membraneless biomolecular condensates governing cell physiology and pathology. Using an archetypical viral protein-RNA condensation model, we determined sequence events that starts with sub-second formation a protomer two RNAs per protein dimer. Association additional RNA molecules to weaker secondary binding sites in this kickstarts crystallization-like assembly molecular condensate. Primary nucleation faster than sum growth, which multistep process. nuclei grow over hundreds seconds into filaments subsequently nanoclusters circa 600 nm diameter. Cryoelectron microscopy reveals internal structure formed by incoming layers made ribonucleoprotein oligomers, reminiscent genome packing nucleocapsid. These progress liquid condensate droplets undergo further partial coalescence yield typical hydrogel-like coacervates may represent scaffold large factory infected cells. Our integrated experimental kinetic investigation exposes rate limiting steps structures along key biological pathway present across life kingdoms.

Язык: Английский

Assembly of SARS-CoV-2 nucleocapsid protein with nucleic acid DOI Creative Commons
Huaying Zhao, Abdullah M. Syed, Mir M. Khalid

и другие.

Nucleic Acids Research, Год журнала: 2024, Номер 52(11), С. 6647 - 6661

Опубликована: Апрель 8, 2024

Abstract The viral genome of SARS-CoV-2 is packaged by the nucleocapsid (N-)protein into ribonucleoprotein particles (RNPs), 38 ± 10 which are contained in each virion. Their architecture has remained unclear due to pleomorphism RNPs, high flexibility N-protein intrinsically disordered regions, and highly multivalent interactions between RNA binding sites both N-terminal (NTD) C-terminal domain (CTD). Here we explore critical interaction motifs RNPs applying a combination biophysical techniques ancestral mutant proteins different nucleic acids an vitro assay for RNP formation, examining protein variants assembly assay. We find that acid-bound dimers oligomerize via recently described protein–protein interface presented transient helix its long linker region NTD CTD. resulting hexameric complexes stabilized protein-nucleic acid establish crosslinks dimeric subunits. Assemblies CTD offering more than one site stem–loop RNA. Our study suggests model where scaffolding at density on followed cooperative multimerization through linker.

Язык: Английский

Процитировано

11
Molecular Matchmakers: How ATP and Small Amphiphilic Molecules Fine-Tune FET Proteins Clusters DOI
Mrityunjoy Kar

bioRxiv (Cold Spring Harbor Laboratory), Год журнала: 2025, Номер unknown

Опубликована: Апрель 16, 2025

Abstract FET (FUS-EWSR1-TAF15) family proteins inherently form mesoscale molecular assemblies, known as clusters, under physiological conditions at concentrations well below the threshold for phase separation. This study demonstrates that adenosine triphosphate (ATP), an amphiphilic molecule and essential cellular metabolite, modulates size of these sub-saturation clusters in a concentration-dependent manner. At low (1-2 mM), ATP acts crosslinker proteins, resulting larger clusters. moderate (5 decreases but stabilizes. high (10 cluster further diminishes. Other molecules, including common hydrotropes like sodium xylene sulfonate, toluene hexanediol, exhibit comparable effects on protein clustering. Notably, cannot be explained solely by hydrotropic or kosmotropic mechanisms; instead, they stem from non-specific interactions between small molecules. The intrinsic chemical properties molecules play crucial role regulating formation concentrations.

Язык: Английский

Процитировано

0
Protein–RNA condensation kinetics via filamentous nanoclusters DOI

Ramón Peralta Martínez,

Araceli Visentín, Mariano Salgueiro

и другие.

Protein Science, Год журнала: 2025, Номер 34(6)

Опубликована: Май 24, 2025

Abstract Protein–RNA phase separation is at the center of membraneless biomolecular condensates governing cell physiology and pathology. Using an archetypical viral protein–RNA condensation model, we determined sequence events that starts with sub‐second formation a protomer two RNAs per protein dimer. Association additional RNA molecules to weaker secondary binding sites in this kickstarts crystallization‐like assembly molecular condensate. Primary nucleation faster than sum growth, which multistep process. nuclei grow over hundreds seconds into filaments subsequently nanoclusters approximately 600 nm diameter. Cryoelectron microscopy reveals internal structure formed by incoming layers made ribonucleoprotein oligomers, reminiscent genome packing nucleocapsid. These progress liquid condensate droplets undergo further partial coalescence yield typical hydrogel‐like coacervates may represent scaffold large factory infected cells. Our integrated experimental kinetic investigation exposes rate‐limiting steps structures along key biological pathway present across life kingdoms.

Язык: Английский

Процитировано

0
Evolution of Virus-like Features and Intrinsically Disordered Regions in Retrotransposon-derived Mammalian Genes DOI Creative Commons
Rachele Cagliani, Diego Forni, Alessandra Mozzi

и другие.

Molecular Biology and Evolution, Год журнала: 2024, Номер 41(8)

Опубликована: Авг. 1, 2024

Abstract Several mammalian genes have originated from the domestication of retrotransposons, selfish mobile elements related to retroviruses. Some proteins encoded by these maintained virus-like features; including self-processing, capsid structure formation, and generation different isoforms through −1 programmed ribosomal frameshifting. Using quantitative approaches in molecular evolution biophysical analyses, we studied 28 retrotransposon-derived genes, with a focus on features. By analyzing rate synonymous substitutions, show that frameshifting mechanism three (PEG10, PNMA3, PNMA5) is conserved across mammals originates alternative proteins. These were targets positive selection primates, one positively selected sites affects B-cell epitope spike domain PNMA5 capsid, finding reminiscent observations infectious viruses. More generally, found vary their intrinsically disordered region content this directly associated evolutionary rates. Most are located regions some them impact protein posttranslational modifications, such as autocleavage phosphorylation. Detailed analyses properties showed preferentially targeted lower conformational entropy. Furthermore, introduces variation binary sequence patterns orthologues, well chain compaction. Our results shed light trajectories unique class suggest novel approach study how characteristics affected evolution.

Язык: Английский

Процитировано

2
SARS-CoV-2 N protein recruits G3BP to double membrane vesicles to promote translation of viral mRNAs DOI Creative Commons
Siwen Long, M. M. Guzyk, Laura Perez Vidakovics

и другие.

Nature Communications, Год журнала: 2024, Номер 15(1)

Опубликована: Дек. 5, 2024

Abstract Ras-GTPase-activating protein SH3-domain-binding proteins (G3BP) are critical for the formation of stress granules (SGs) through their RNA- and ribosome-binding properties. SARS-CoV-2 nucleocapsid (N) exhibits strong binding affinity G3BP inhibits infection-induced SG soon after infection. To study impact G3BP-N interaction on viral replication pathogenesis in detail, we generated a mutant (RATA) that specifically lacks G3BP-binding motif N protein. RATA triggers stronger more persistent response infected cells, showing reduced across various cell lines, greatly K18-hACE2 transgenic mice. At early times infection, WT strongly colocalise with dsRNA non-structural 3 (nsp3), component pore complex double membrane vesicles (DMVs) from which nascent RNA emerges. Furthermore, complexes promote highly localized translation mRNAs immediate vicinity DMVs thus contribute to efficient gene expression replication. In contrast, is absent cells less efficient. This work provides fuller understanding multifunctional roles

Язык: Английский

Процитировано

1
A core network in the SARS-CoV-2 nucleocapsid NTD mediates structural integrity and selective RNA-binding DOI Creative Commons
Karthikeyan Dhamotharan, Sophie Marianne Korn, Anna Wacker

и другие.

Nature Communications, Год журнала: 2024, Номер 15(1)

Опубликована: Дек. 9, 2024

Abstract The SARS-CoV-2 nucleocapsid protein is indispensable for viral RNA genome processing. Although the N-terminal domain (NTD) suggested to mediate specific RNA-interactions, high-resolution structures with are still lacking. Available hybrid of NTD ssRNA and dsRNA provide valuable insights; however, precise mechanism complex formation remains elusive. Similarly, molecular impact mutations that have emerged since 2019 has not yet been fully explored. Using crystallography solution NMR, we investigate how influence structural integrity RNA-binding. We find both features rely on a core network residues conserved in Betacoronaviruses , crucial stability communication among flexible loop-regions facilitate RNA-recognition. Our comprehensive analysis demonstrates contacts within this guide selective RNA-interactions. propose renders evolutionarily robust plasticity its versatile processing roles.

Язык: Английский

Процитировано

1
Protein-RNA condensation kinetics via filamentous nanoclusters DOI Creative Commons

Ramón Peralta Martínez,

Araceli Visentín, Mariano Salgueiro

и другие.

bioRxiv (Cold Spring Harbor Laboratory), Год журнала: 2024, Номер unknown

Опубликована: Ноя. 8, 2024

Abstract Protein-RNA phase separation is at the center of membraneless biomolecular condensates governing cell physiology and pathology. Using an archetypical viral protein-RNA condensation model, we determined sequence events that starts with sub-second formation a protomer two RNAs per protein dimer. Association additional RNA molecules to weaker secondary binding sites in this kickstarts crystallization-like assembly molecular condensate. Primary nucleation faster than sum growth, which multistep process. nuclei grow over hundreds seconds into filaments subsequently nanoclusters circa 600 nm diameter. Cryoelectron microscopy reveals internal structure formed by incoming layers made ribonucleoprotein oligomers, reminiscent genome packing nucleocapsid. These progress liquid condensate droplets undergo further partial coalescence yield typical hydrogel-like coacervates may represent scaffold large factory infected cells. Our integrated experimental kinetic investigation exposes rate limiting steps structures along key biological pathway present across life kingdoms.

Язык: Английский

Процитировано

0