Post-transcriptional regulation of the endoplasmic reticulum stress response in Cryptococcus neoformans

Cryptococcus neoformans is one of a small number of fungi able to transition from environmental temperatures to human core body temperature. Adaptation to human body temperature allows this fungus to disseminate and invade the brain where it causes meningoencephalitis. In C. neoformans , deletion of the RNA-binding protein Puf4, or the deadenylase Ccr4, results in delayed growth at 37°C, suggesting Puf4 and Ccr4 are required for the host temperature adaptation process.;In the document that follows, we characterize a role for Puf4 and Ccr4 in the post-transcriptional regulation of the ER stress response during growth at 37°C. We demonstrate that in C. neoformans, growth at 37°C induces the ER stress response, also termed the Unfolded Protein Response (UPR). Once triggered, UPR induction occurs through the unconventional splicing of an mRNA, called HXL1 in C. neoformans , which encodes the UPR transcription factor. Splicing allows for the translation of the active UPR transcription factor that is responsible for promoting the expression of UPR genes. Our studies describe a role for Puf4 in regulating the rate of HXL1 unconventional splicing, with delays in splicing resulting in delayed induction of UPR transcripts. Additionally, both Puf4 and Ccr4 are required for the decay of ER stress response transcripts following the adaptation of the cell to growth at 37°C, when the transcripts are no longer necessary. However, Puf4 appears to primarily regulate the HXL1 transcript, whereas Ccr4 mediates the decay of a multitude of UPR transcripts following host temperature adaptation.;Taken as a whole, our studies suggest post-transcriptional modulation of UPR transcripts influences the rate and kinetics of UPR mRNA induction, with deletion of Puf4 or Ccr4 skewing UPR transcript levels. We postulate that in the puf4Delta and ccr4Delta mutants, the changes in UPR transcript levels may contribute to some of the phenotypes observed in these strains including cell wall integrity defects, resistance to the ER stress inducing drug tunicamycin, and sensitivity to growth at human core body temperature. All of which are dependent on a properly functioning UPR. Our research is the first to characterize an RNA-binding protein, Puf4, as regulating the unconventional splicing of the mRNA encoding the UPR transcription factor in any species. We describe a divergent role for Puf4 compared to Puf4p in Saccharomyces cerevisiae, and a novel convergence in the evolution of the UPR signaling pathway and post-transcriptional regulation of mRNA by PUF proteins in C. neoformans..