Post-transcriptional gene regulation contributes to host temperature adaptation in the pathogenic fungus cryptococcus neoformans

Tightly controlled changes in gene expression are required for proper reprogramming of cellular processes that promote adaptation to changes in the environment. The ability to adapt to human host temperature is a unique trait that separates the few deep-infectious fungal pathogens from the millions of existing fungal species. The objective of these studies was to understand the gene expression changes that occur in Cryptococcus neoformans in response to host temperature, and investigate post-transcriptional mechanisms that contribute to executing these adaptation-promoting changes. In the first part of these studies, we revealed that specific classes of functionally related stress response transcripts undergo induction concomitantly with repression of the abundant ribosomal protein (RP) transcripts, and that these gene expression changes are transient. Stability analyses demonstrated that temporally regulated enhanced destabilization of these classes of transcripts, or decay regulons, controlled the intensity and duration of these responses. Enhanced degradation was dependent on the cellular deadenylase, Ccr4. Specificity in the classes of transcripts as well as the timing of degradation was dependent on the dissociable RNA polymerase II subunit, Rpb4, suggesting that during stress mRNA synthesis and degradation are coupled. Further, we revealed that distinct signaling modules activate the decay regulons. Both rpb4Delta and ccr4Delta mutant strains exhibited sensitivity to growth at host temperature and are attenuated for virulence in mouse models of cryptococcosis. These studies revealed an elegant, tightly controlled system of post-transcriptional regulation that significantly contributes to fine-tuning gene expression changes that promote host temperature adaptation.;In the second part of our study, we investigated the downstream effects of gene expression changes on translation during host temperature stress to understand the importance of these changes in cellular reprogramming. Polysome profiles revealed that the translational state of the cells is not affected by host temperature stress in the wild type, but instead, changes in gene expression promote reprogramming of the actively translating pool of mRNA. Specifically, repression of the RP transcripts allows the induced stress-specific transcripts access to the translational machinery. This translational reprogramming was dependent on mRNA degradation, as changes in the actively translating pool were less efficient in decay mutants. We specifically reveal that Ccr4-dependent degradation serves to balance the levels of cellular mRNA during stress in order to avoid saturation of the translational machinery. Preliminary assessment of the changes in the actively translating pools of mRNA during host temperature adaptation by RNA sequencing supported our findings in regard to changes in RP and ER stress transcripts, and has revealed additional interesting changes that will be pursued in the future.;In the third part of our studies, we sought to identify the cis acting element and specific RNA binding protein that mediates the degradation of RP transcripts in order to elaborate our understanding of this important post-transcriptional mechanism. Using motif-based sequence analysis we identified a shared cis element in the 3'UTRs of RP transcripts that demonstrated specific protein binding activity by electrophoretic mobility shift assays. Affinity chromatography and mass spectrometry revealed that the zinc knuckle protein, Gis2, specifically interacts with this element. While the shared cis element was not necessary for enhanced mRNA degradation of RP transcripts, the inability to knock out GIS2 suggests that this protein may an important component of regulating ribosomal protein expression. Based on our data, we conclude that the precise control of post-transcriptional regulation plays a major role in the ability of C. neoformans to reprogram cellular processes in response to stress, and contributes to proper gene expression necessary for maintaining homeostasis and pathogenicity.