Post-transcriptional regulation of fungal cell walls by RNA-binding proteins and untranslated regions

Abstract

Cells grow and divide while maintaining spatial organisation, by controlling gene expression and subcellular RNA and protein localisation. Localised control of protein synthesis is important for establishment of cellular asymmetries, and for cell wall biogenesis at the growth tip leading to directional growth. Ssd1 is a conserved RNA-binding protein (RBP), required for tolerance of heat and cell wall stress in Saccharomyces cerevisiae and for virulence in Candida albicans and Candida glabrata. I hypothesize that Ssd1 is important for localised control of cell wall protein synthesis, thus contributing to directional growth and virulence.

Recent data from the Wallace lab show Ssd1 binding near the start codons of mRNAs encoding specific cell wall proteins. My research tests the hypothesis that Ssd1, alongside other RBPs of interest, control localised translation of target cell wall mRNAs near sites of cell wall synthesis. I employ single molecule fluorescence in situ hybridization (smFISH) to understand regulation of target mRNAs in S. cerevisiae. Using microscopy and flow cytometry, I also investigate the effect of Ssd1 and untranslated regions (UTRs) on the production and localisation of cell wall proteins fused to fluorescent tags. Lastly, I study conservation of Ssd1 molecular function in pathogenic fungi through genetic complementation with Ssd1 homologues from pathogens into S. cerevisiae.

I do not find clear evidence to support Ssd1 affecting protein localisation, however SSD1 deletion leads to protein overproduction. Therefore, my results support the hypothesis that Ssd1 represses the translation of cell wall proteins to promote localised protein synthesis. My work shows that RNA targets of Ssd1 localise in a cell-cycle dependent manner. These findings in budding yeast can generate insight into post-transcriptional control of cell walls in pathogenic fungi, which pose major threats to human and crop health and rely on homologous systems of mRNA transport for growth and virulence.