Mod5 mediates a molecular trade-off between transcriptional and translational regulation and antifungal resistance.
Increasing antifungal drug resistance is a major concern associated with human fungal infections. Genetic mutation and epimutation mechanisms are clearly linked to resistance in some fungi, but few studies have investigated RNA modifications (the epitranscriptome) as a mediator of resistance. Here, deletion of the Aspergillus fumigatus tRNA-modifying isopentenyl transferase ortholog, Mod5, led to altered growth and stress response, but also unexpected resistance against the antifungal drug 5-fluorocytosine (5-FC). Simultaneous profiling of the transcriptome and proteome of 5-FC-stressed wild-type and ?mod5 strains revealed a similar general adaptation to stress; however, the knockout displayed elevated expression of cross-pathway control (CPC) genes. Quantification of CPC transcription factor cpcA and client gene argB showed premature CPC activation in ?mod5, which was further increased upon treatment with 5-FC. By associating codon usage patterns with proteomics abundances, we observed the number of tRNATyrG?A-decoded codons to negatively correlate with protein abundance in the knockout, indicative of modification-tunable transcripts. Overexpression of tRNATyrG?A in the ?mod5 strain rescued select phenotypes but, surprisingly, failed to reverse 5-FC resistance. Investigation of the purported tRNA gene-mediated silencing function of Mod5 uncovered a negative correlation between tRNA proximity and gene induction under normal growth, suggesting that tRNA gene-mediated silencing is active in A. fumigatus. In conclusion, 5-FC resistance in the absence of bifunctional Mod5 likely originates from multifaceted transcriptional and translational changes that skew the fungus towards starvation responses over optimal growth, potentially offering a mechanism reliant on RNA modification and tRNA gene-mediated silencing capable of facilitating transient antifungal resistance. Overall design: Comparative gene expression profiling analysis of poly(A) RNA-seq data for ??mod5 knockouts of Aspergillus fumigatus mycelium left unstressed for 24 hours in Aspergillus minimal media or grown for 16 hours unstressed followed by 8 hours stressed with 5-fluorocytosine.