The fungal nucleolus is responsible for coordinating ribosome biogenesis with the physiological demand for protein synthesis. In Aspergillus fumigatus the requirement for ribosomes is particularly high during conidial germination and hyphal growth, suggesting that ribosome synthesis would be an effective target for both prophylactic and therapeutic intervention. We have identified a nucleolar protein of unknown function that is highly conserved among fungi. As a first step to validating the A. fumigatus ortholog as an antifungal target we have defined the functional role of the corresponding protein in Saccharomyces cerevisiae, Cgr1p. Loss of Cgr1p functional activity was accomplished in two mutant strains: a conditional depletion strain in which CGR1 transcription was placed under the control of a tetracycline-repressible promoter, and a truncation strain in which the expressed Cgr1p was shortened at the c-terminus by 40 amino acids. Impaired Cgr1 function was accompanied by a reduction in growth rate and hypersensitivity to translational inhibitors. Polysome analyses of cgr1 mutants revealed an imbalanced ratio of free 40S subunits to 60S subunits, a decrease in 80S monosomes and accumulation of half-mer polysomes. Pulse-chase labeling demonstrated that pre-rRNA processing was defective, resulting in abnormal accumulation of the 35S, 27S and 7S pre-rRNAs and delayed production of the mature 25S and 5.8S rRNAs. The synthesis of the 18S and 5S rRNAs was unaffected. These studies establish a clear role for Cgr1p in fungal ribosome synthesis, thus providing a rational basis for future studies into how this protein impacts ribosome synthesis and virulence in A. fumigatus.
Full conference title:
American Society for Microbiology General Meeting
- ASM 102nd (2002)