An official website of the United States government
Abstract The Nucleocapsid protein (N) of SARS-CoV-2 plays a critical role in the viral lifecycle by regulating RNA replication and by packaging the viral genome. N and RNA phase separate to form condensates that may be important for these functions. Both functions occur at membrane surfaces, but how N toggles between these two membrane-associated functional states is unclear. Here, we reveal that phosphorylation switches how N condensates interact with membranes, in part by modulating condensate material properties. Our studies also show that phosphorylation alters N’s interaction with viral membrane proteins. We gain mechanistic insight through structural analysis and molecular simulations, which suggest phosphorylation induces a conformational change in N that softens condensate material properties. Together, our findings identify membrane association as a key feature of N condensates and provide mechanistic insights into the regulatory role of phosphorylation. Understanding this mechanism suggests potential therapeutic targets for COVID infection.
more »
« less
RNA encodes physical information
Abstract Most amino acids are encoded by multiple codons, making the genetic code degenerate. Synonymous mutations affect protein translation and folding, but their impact on RNA itself is often neglected. We developed a genetic algorithm that introduces synonymous mutations to control the diversity of structures sampled by an mRNA. The behavior of the designed mRNAs reveals a physical code layered in the genetic code. We find that mRNA conformational heterogeneity directs physical properties and functional outputs of RNA-protein complexes and biomolecular condensates. The role of structure and disorder of proteins in biomolecular condensates is well appreciated, but we find that RNA conformational heterogeneity is equally important. This feature of RNA enables both evolution and engineers to build cellular structures with specific material and responsive properties.
more »
« less
- Award ID(s):
- 2044895
- PAR ID:
- 10667656
- Publisher / Repository:
- bioRxiv
- Date Published:
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
More Like this
-
-
Cimarelli, Andrea (Ed.)The Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) infection causes Coronavirus Disease 2019 (COVID-19), a pandemic that seriously threatens global health. SARS-CoV-2 propagates by packaging its RNA genome into membrane enclosures in host cells. The packaging of the viral genome into the nascent virion is mediated by the nucleocapsid (N) protein, but the underlying mechanism remains unclear. Here, we show that the N protein forms biomolecular condensates with viral genomic RNA both in vitro and in mammalian cells. While the N protein forms spherical assemblies with homopolymeric RNA substrates that do not form base pairing interactions, it forms asymmetric condensates with viral RNA strands. Cross-linking mass spectrometry (CLMS) identified a region that drives interactions between N proteins in condensates, and deletion of this region disrupts phase separation. We also identified small molecules that alter the size and shape of N protein condensates and inhibit the proliferation of SARS-CoV-2 in infected cells. These results suggest that the N protein may utilize biomolecular condensation to package the SARS-CoV-2 RNA genome into a viral particle.more » « less
-
Abstract Scaffold proteins play crucial roles in subcellular organization and function. In many organisms, proteins with multiple Tudor domains are required for the assembly of membraneless RNA-protein organelles (germ granules) in germ cells. Tudor domains are protein-protein interaction modules which bind to methylated polypeptides.DrosophilaTudor protein contains eleven Tudor domains, which is the highest number known in a single protein. The role of each of these domains in germ cell formation has not been systematically tested and it is not clear if some domains are functionally redundant. Using CRISPR methodology, we generated mutations in several uncharacterized Tudor domains and showed that they all caused defects in germ cell formation. Mutations in individual domains affected Tudor protein differently causing reduction in protein levels, defects in subcellular localization and in the assembly of germ granules. Our data suggest that multiple domains of Tudor protein are all needed for efficient germ cell formation highlighting the rational for keeping many Tudor domains in protein scaffolds of biomolecular condensates inDrosophilaand other organisms.more » « less
-
Abstract Single-stranded RNA molecules can form intramolecular bonds between nucleotides to create secondary structures. These structures can have phenotypic effects, meaning mutations that alter secondary structure may be subject to natural selection. Here, we examined the population genetics of these mutations within Arabidopsis thaliana genes. We began by identifying derived SNPs with the potential to alter secondary structures within coding regions, using a combination of computational prediction and empirical data analysis. We identified 8,469 such polymorphisms, representing a small portion (∼0.024%) of sites within transcribed genes. We examined nucleotide diversity and allele frequencies of these “pair-changing mutations” (pcM) in 1,001 A. thaliana genomes. The pcM SNPs at synonymous sites had a 13.4% reduction in nucleotide diversity relative to non-pcM SNPs at synonymous sites and were found at lower allele frequencies. We used demographic modeling to estimate selection coefficients, finding selection against pcMs in 5′ and 3′ untranslated regions. Previous work has shown that some pcMs affect gene expression in a temperature-dependent matter. We explored associations on a genome-wide scale, finding that pcMs existed at higher population frequencies in colder environments, but so did non-PCM alleles. Derived pcM mutations had a small but significant relationship with gene expression; transcript abundance for pcM-containing alleles had an average reduction in expression of ∼4% relative to alleles with conserved ancestral secondary structure. Overall, we document selection against derived pcMs in untranslated regions but find limited evidence for selection against derived pcMs at synonymous sites.more » « less
-
Biomolecular condensates mediate diverse and essential cellular functions by compartmentalizing biochemical pathways. Many condensates have internal subdomains with distinct compositional identities. A major challenge lies in dissecting the multicomponent logic that relates biomolecular features to emergent condensate organization. Nuclear paraspeckles are paradigmatic examples of multidomain condensates, comprising core and shell layers with distinct compositions that are scaffolded by the lncRNA NEAT1, which spans both layers. A prevailing model of paraspeckle assembly proposes that core proteins bind directly and specifically to core-associated NEAT1 domains. Combining informatics and biochemistry, we unexpectedly find that the essential core proteins FUS and NONO bind and condense preferentially with shell-associated NEAT1 domains. The shell protein TDP-43 exhibits similar NEAT1 domain preferences on its own but forms surfactant-like shell layers around core protein-driven condensates when both are present. Together, experiments and physics-based simulations suggest that competitive RNA binding and immiscibility between core and shell proteins order paraspeckle layers. More generally, we propose that subcondensate organization can spontaneously arise from a balance of collaborative and competitive protein binding to the same domains of a lncRNA.more » « less
- Free Publicly Accessible Full Text
-
Accepted Manuscript
- Posted Content:
- https://doi.org/10.1101/2024.12.11.627970
