Biology subjects

Danecka, W.

Publications and source records attributed to Danecka, W..

5 recordsLinked to original sources

bioRxiv2026

The fungal RNA-binding protein Ssd1 represses Sun4 protein abundance through recognition of 5' UTR structural and sequence elements

Regulation of protein abundance allows fungi to adapt to changing environments, regulate their growth and morphology, and react to external stresses. Ssd1 is a fungal RNA binding protein that binds mRNAs encoding cell wall remodelling proteins and regulates cell wall biogenesis. Ssd1 binding sites (SBSs) have been described by computational and biochemical analyses, but how Ssd1 recognises these sites and how that relates to regulation of protein abundance was less clear. Here, a co-crystal structure of Saccharomyces cerevisiae Ssd1 with an SBS reveals core determinants of recognition, while fluorescent reporters of Sun4, an Ssd1-regulated cell wall protein, were used to characterise structure- guided mutations. We find that Ssd1 has an extensive RNA binding site that recognises two elements of the SBS: an upstream element that forms a structural motif, and an element containing tandem CNYU sequences that engages Ssd1 in base-specific recognition. Mutations to the Ssd1 RNA binding surface prevent Ssd1-dependent repression of fluorescent reporters and show strong phenotypes in assays of cell wall stress resistance and genetic interactions with the Cbk1 kinase. Loss of repression is also observed if both SUN4 SBSs are altered. However, the presence of one SBS in the SUN4 5' untranslated region is sufficient to confer Ssd1-dependent suppression of protein abundance. Our work confirms that RNA binding is a core function of Ssd1 and is likely to inform functional analyses in fungi beyond S. cerevisiae, where Ssd1 orthologs have been identified as virulence factors in several fungal pathogens.

molecular biology↗

bioRxiv2025

Antifungal exposure can enhance Candida glabrata pathogenesis

Azole antifungal drugs directly inhibit lanosterol 14-[a]-demethylase and indirectly affect the expression of metabolic, transmembrane transporter, and cell wall organization genes in fungal pathogens. It is not known how these indirect azole effects depend on dose, timing, and specific azole used, or how they influence host interactions. Candida glabrata (recently renamed Nakaseomyces glabratus) is the second leading cause of candidiasis, and clinical strains have high rates of intrinsic resistance to azoles. We investigated the early responses of reference strains BG2 and CBS138 to sub-inhibitory doses of fluconazole and voriconazole, and particularly, how these responses affect host-pathogen interactions. Cell wall profiling and transcriptomic data revealed highly similar responses for each strain to both azoles, including the upregulation of several virulence factors, such as yapsins. We also observed significant increases in CBS138 survival in macrophages and increased virulence in Galleria mellonella after voriconazole exposure. Using a combination of pharmacological inhibition of calcium ion channels and deletion strains, we determined that voriconazole-enhanced virulence requires a yapsin protease, YPS1, and is regulated via the calcineurin pathway and the cell wall integrity pathway, both of which regulate YPS1 expression. We also observed that voriconazole treatment significantly reduced the virulence of the bck1{Delta} strain in G. mellonella, suggesting that inhibitors of the cell wall integrity pathway might potentiate azole activity by improving susceptibility to host killing. Our study provides new insight into short-term azole adaptation in C. glabrata, and importantly demonstrates that sub-inhibitory azole exposure can induce virulence factors and alter fungal pathogenesis. Article summaryAntifungal drugs indirectly affect essential fungal cell processes, but we lack an understanding of how drug-induced changes affect fungal pathogenesis. We investigated how Candida glabrata adapts when exposed to azole drugs in terms of cell wall and transcriptional changes. Reference strains had similar transcriptional changes in response to azoles, but azole-treated CBS138 survived better in immune cells and caused more host death than untreated cells, suggesting that short-term azole treatment can significantly affect pathogenesis. Voriconazole-enhanced disease requires the calcineurin and cell wall integrity pathways and the virulence factor, YPS1, but could be blocked by a calcium ion channel inhibitor. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=110 SRC="FIGDIR/small/667834v1_ufig1.gif" ALT="Figure 1"> View larger version (29K): org.highwire.dtl.DTLVardef@157eaforg.highwire.dtl.DTLVardef@13050c0org.highwire.dtl.DTLVardef@10593d0org.highwire.dtl.DTLVardef@12b663e_HPS_FORMAT_FIGEXP M_FIG C_FIG

microbiology↗

bioRxiv2023

The type of carbon source not the growth rate it supports can determine diauxie

How cells choose between potential carbon sources is a classic example of cellular decision-making, and we know that many organisms prioritise glucose. Yet there has been little investigation of whether other sugars are also preferred, blinkering our view of carbon sensing. Here we study eukaryotic budding yeast and its growth on mixtures of palatinose, an isomer of sucrose, with other sugars. We find that yeast prioritise galactose over palatinose, but not sucrose or fructose, despite all three of these sugars being able to support faster growth than palatinose. Our results therefore disfavour carbon flux-sensing as the sole mechanism. By using genetic perturbations and transcriptomics, we show that repression is active and through Gal4, the master regulator of the GAL regulon. Cells enforce their preference for galactose over palatinose by preventing runaway positive feedback in the MAL regulon, whose genes enable palatinose catabolism. They do so both by repressing MAL11, the gene encoding the palatinose transporter, and by first expressing the isomaltases, IMA1 and IMA5, which cleave palatinose and so prevent its intracellular concentration becoming enough to induce further MAL expression. Our results demonstrate that budding yeast actively maintain a preference for carbon sources other than glucose and that such preferences have been selected by more than differences in growth rates. They imply that carbon-sensing strategies even in unicellular organisms are more complex than previously thought.

systems biology↗

bioRxiv2023

Extensible benchmarking of methods that identify and quantify polyadenylation sites from RNA-seq data

The tremendous rate with which data is generated and analysis methods emerge makes it increasingly difficult to keep track of their domain of applicability, assumptions, and limitations and consequently, of the efficacy and precision with which they solve specific tasks. Therefore, there is an increasing need for benchmarks, and for the provision of infrastructure for continuous method evaluation. APAeval is an international community effort, organized by the RNA Society in 2021, to benchmark tools for the identification and quantification of the usage of alternative polyadenylation (APA) sites from short-read, bulk RNA-sequencing (RNA-seq) data. Here, we reviewed 17 tools and benchmarked eight on their ability to perform APA identification and quantification, using a comprehensive set of RNA-seq experiments comprising real, synthetic, and matched 3'-end sequencing data. To support continuous benchmarking, we have incorporated the results into the OpenEBench online platform, which allows for seamless extension of the set of methods, metrics, and challenges. We envisage that our analyses will assist researchers in selecting the appropriate tools for their studies. Furthermore, the containers and reproducible workflows generated in the course of this project can be seamlessly deployed and extended in the future to evaluate new methods or datasets.

bioinformatics↗

bioRxiv2021

Probing 3'UTRs as modular regulators of gene expression

Genes are commonly abstracted into a coding sequence and cis-regulatory elements (CREs), such as promoters and terminators, and short sequence motifs within these regions. Modern cloning techniques allow easy assembly of synthetic genetic constructs from discrete cis-regulatory modules. However, it is unclear how much the contributions of CREs to gene expression depend on other CREs in the host gene. Using budding yeast, we probe the extent of composability, or independent effects, of distinct CREs. We confirm that the quantitative effect of a terminator on gene expression depends on both promoter and coding sequence. We then explore whether individual cisregulatory motifs within terminator regions display similar context dependence, using transcriptomewide datasets of mRNA decay. To test the extent of composability, we construct reporter genes consisting of combinations of motifs within various terminator contexts, paired with different promoters. Our results show that the effect of a motif on RNA abundance depends both on its host terminator, and also on the associated promoter sequence. This emphasises the need for improved motif inference that includes both local and global context effects, which in turn could aid in the accurate use of CREs for the engineering of synthetic genetic constructs.

systems biology↗