Halofuginone triggers a transcriptional program centered on ribosome biogenesis and function in honey bees

We previously found that pharmacological inhibition of prolyl-tRNA synthetase by halofuginone has potent activity against Nosema ceranae, an important pathogen of honey bees. However, we also observed that prolyl-tRNA synthetase inhibition is toxic to bees, suggesting further work is necessary to make this a feasible therapeutic strategy. As expected, we found that pharmacological inhibition of prolyl-tRNA synthetase activity resulted in robust induction of select canonical ATF4 target genes in honey bees. However, our understanding of this and other cellular stress responses in general in honey bees is incomplete. Thus, we used RNAseq to identify novel changes in gene expression after halofuginone treatment and observed induction of genes involved in ribosome biogenesis, translation, tRNA synthesis, and ribosome-associated quality control (RQC). These results suggest that halofuginone, potentially acting through the Integrated Stress Response (ISR), promotes a transcriptional response to ribosome functional impairment in honey bees rather than the response designed to oppose amino acid limitation, which has been observed in other organisms after ISR induction. In support of this idea, we found that cycloheximide (CHX) administration also induced all tested target genes, indicating that this gene expression program could be induced by ribosome stalling in addition to tRNA synthetase inhibition. Only a subset of halofuginone-induced genes was upregulated by Unfolded Protein Response (UPR) induction, suggesting that mode of activation and cross-talk with other cellular signaling pathways significantly influence ISR function and cellular response to its activation. Future work will focus on understanding how the apparently divergent transcriptional output of the ISR in honey bees impacts the health and disease of this important pollinator species.