Biology subjects

Cole, S. J.

Publications and source records attributed to Cole, S. J..

5 recordsLinked to original sources

bioRxiv2026

Sequence and structure of protein binding sites in RNA impact biomolecular condensates

Biomolecular condensates are central to subcellular compartmentalization. Although many condensates contain and regulate RNA, research has primarily focused on protein interactions. Here, we investigate RNA-protein interactions underlying cell cycle-regulating condensates in the multinucleate fungus Ashbya gossypii. These condensates form through interactions between G1 cyclin mRNA CLN3 and RNA-binding protein Whi3, which was predicted by homology to recognize a five-nucleotide motif repeated within the transcript. Natural variation in motif number in Ashbya strains led us to hypothesize that binding site valence may influence condensate properties. Using unbiased binding assays, we determined the preference of Ashbya Whi3 protein for specific primary RNA sequence and mutated individual Whi3-binding sites within CLN3 mRNA. Mutants exhibited distinct condensate properties despite having the same valence in terms of binding site number. Mutations altered the saturation concentration (Csat) and dense phase concentration of RNA and protein in cell-free reconstitution experiments. A subset of mutants, showed reduced number of condensates and deregulation of the cell cycle in cells. We also find that enhanced availability of single-stranded RNA can compensate for loss of binding sites. Together, these data indicate that differences in RNA protein binding-site context and not simply valence plays a critical role in determining condensate properties.

cell biology↗

bioRxiv2025

SARS-CoV-2 cellular coinfection is limited by superinfection exclusion.

The coinfection of individual cells is a requirement for exchange between two or more virus genomes, which is a major mechanism driving virus evolution. Coinfection is restricted by a mechanism known as superinfection exclusion (SIE), which prohibits the infection of a previously infected cell by a related virus after a period of time. SIE regulates coinfection for many different viruses, but its relevance to the infection of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) was unknown. In this study, we investigated this using a pair of SARS-CoV-2 variant viruses encoding distinct fluorescent reporter proteins. We show for the first time that SARS-CoV-2 coinfection of individual cells is limited temporally by SIE. We defined the kinetics of the onset of SIE for SARS-CoV-2 in this system, showing that the potential for coinfection starts to diminish within the first hour of primary infection, and then falls exponentially as the time between the two infection events is increased. We then asked how these kinetics would affect the potential for coinfection with viruses during a spreading infection. We used plaque assays to model the localised spread of SARS-CoV-2 observed in infected tissue, and showed that the kinetics of SIE restrict coinfection, and therefore sites of possible genetic exchange, to a small interface of infected cells between spreading viral infections. This indicates that SIE, by reducing the likelihood of coinfection of cells, likely reduces the opportunities for genetic exchange between different strains of SARS-CoV-2 and therefore is an underappreciated factor in shaping SARS-CoV-2 evolution.

microbiology↗

bioRxiv2024

Condensates act as translation hubs to coordinate multinucleate cell growth

Coordination between growth and nuclear division is a common cell feature. In some syncytia, nuclei divide asynchronously throughout the cell but growth occurs only at discrete locations, raising the question how the processes are locally regulated and globally coordinated. In the syncytial fungus Ashbya gossypii, both cell-cycle progression and hyphal elongation require condensates formed by the protein Whi3 in complex with distinct mRNA species. We show that Whi3 condensates are enriched for translation regulators and are associated with local, spatially patterned translation of specific target RNAs near nuclei and growth sites. Whi3-RNA condensates can both promote and repress mRNA translation in an RNA- and condensate size-dependent manner in vitro. Condensate-interfaces are sites of translation, tunable by condensate composition, RNA valency and protein charge-state in vitro. Together, these data suggest that Whi3 condensates can generate a continuum of translation states that vary depending on the subcellular location and resident RNA sequences.

cell biology↗

bioRxiv2024

RNA encodes physical information

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.

biophysics↗

bioRxiv2023

Characterization of the Entner-Douderoff Pathway in Pseudomonas aeruginosa Catheter-associated Urinary Tract Infections

Pseudomonas aeruginosa is an opportunistic nosocomial pathogen responsible for catheter-associated urinary tract infections (CAUTI). In a murine model of P. aeruginosa CAUTI, we previously demonstrated that urea within urine suppresses quorum sensing and induces the Entner-Douderoff (E-D) pathway. The E-D pathway consists of the genes zwf, pgl, edd, and eda. Zwf and Pgl convert glucose-6-phosphate into 6-phosphogluconate. Edd hydrolyzes 6-phosphogluconate to 2-keto-3-deoxy-6-phosphogluconate (KDPG). Finally, Eda cleaves KDPG to glyceraldehyde-3-phosphate and pyruvate, which enters the citric acid cycle. Here, we generated in-frame E-D mutants in strain PA14 and assessed their growth phenotypes on chemically defined media. These E-D mutants have a growth defect when grown on glucose or gluconate as sole carbon source which are similar to results previously reported for PAO1 mutants lacking E-D genes. RNA-sequencing following short exposure to urine revealed minimal gene regulation differences compared to the wild type. In a murine CAUTI model, virulence testing of E-D mutants revealed that two mutants lacking zwf and pgl showed minor fitness defects. Infection with the {Delta}pgl strain exhibited a 20% increase in host survival, and the {Delta}zwf strain displayed decreased colonization of the catheter and kidneys. Consequently, our findings suggest that the E-D pathway in P. aeruginosa is dispensable in this model of CAUTI. ImportancePrior studies have shown that the Entner-Douderoff pathway is up-regulated when Pseudomonas aeruginosa is grown in urine. Pseudomonads use the Entner-Douderoff pathway to metabolize glucose instead of glycolysis which led us to ask whether this pathway is required for urinary tract infection. Here, single-deletion mutants of each gene in the pathway were tested for growth on chemically defined media with single-carbon sources as well as complex media. The effect of each mutant on global gene expression in laboratory media and urine was characterized. The virulence of these mutants in a murine model of catheter-associated urinary tract infection revealed that these mutants had similar levels of colonization indicating that glucose is not the primary carbon source utilized in the urinary tract.

microbiology↗