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, required for tolerance of heat and cell wall stress in Saccharomyces cerevisiae and for virulence in Candida albicans and Candida glabrata. We hypothesize that Ssd1 is important for localised control of cell wall protein synthesis, thus contributing to directional growth and virulence.
Recent data from our lab show Ssd1 binding near the start codons of mRNAs encoding specific cell wall proteins. We test the hypothesis that Ssd1 controls localised translation of target cell wall mRNAs near sites of cell wall synthesis. We employ single molecule fluorescence in situ hybridisation (smFISH) to understand Ssd1 regulation of target mRNAs in S. cerevisiae, and ribosome profiling to quantify translation per mRNA in wild-type and ∆ssd1 cells. Using microscopy and flow cytometry, we also investigate the effect of Ssd1 on the production and localisation of cell wall proteins fused to fluorescent tags. Lastly, we study conservation of Ssd1 molecular function in pathogenic fungi through genetic complementation with Ssd1 homologues from pathogens into S. cerevisiae.
Our results support the hypothesis that Ssd1 represses the translation of cell wall proteins to promote localised protein synthesis. We show that RNA targets of Ssd1 localise in a cell-cycle dependent manner. However, we do not find clear evidence to support effects of Ssd1 on mRNA localisation, suggesting involvement of other RNA-binding proteins. Our 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 analogous systems of mRNA transport for growth and virulence.