Puf4 Regulates both Splicing and Decay of HXL1 mRNA Encoding the Unfolded Protein Response Transcription Factor in Cryptococcus neoformans

Abstract

The endoplasmic reticulum (ER) responds to errors in protein folding or processing by induction of the unfolded protein response (UPR). During conditions of ER stress, unconventional splicing of an mRNA encoding the UPR-responsive transcription factor occurs at the ER surface, resulting in activation of the UPR. UPR activation is necessary for adaptation to ER stress and for the pathogenic fungus Cryptococcus neoformans is an absolute requirement for temperature adaptation and virulence. In this study, we have determined that C. neoformans has co-opted a conserved PUF RNA binding protein to regulate the posttranscriptional processing of the HXL1 mRNA encoding the UPR transcription factor. PUF elements were identified in both the 5= and 3= untranslated regions of the HXL1 transcript, and both elements bound Puf4. Deletion of PUF4 resulted in delayed unconventional splicing of HXL1 mRNA and delayed induction of Hxl1 target genes. In addition, the HXL1 transcript was stabilized in the absence of Puf4. The puf4⌬ mutant exhibited temperature sensitivity but was as virulent as the wild type, despite a reduction in fungal burden in the brains of infected mice. Our results reveal a novel regulatory role in which a PUF protein influences the unconventional splicing of the mRNA encoding the UPR-responsive transcription factor. These data suggest a unique role for a PUF protein in controlling UPR kinetics via the posttranscriptional regulation of the mRNA encoding the UPR transcription factor Hxl1. Cryptococcus neoformans is one of the few fungal species that has evolved to successfully transition from its environmental niche to cause deep infections in humans, which requires adaptation to the mammalian core temperature. The mammalian host temperature exerts endoplasmic reticulum (ER) stress on C. neoformans, leading to a transient induction of the unfolded protein response (UPR) during temperature adaptation (1). The loss of UPR induction through deletion of the stress response mediators IRE1 and HXL1 impairs virulence and temperature adaptation (2). Likewise, prolonged ER stress resulting from the loss of mRNA degradation in a ccr4⌬ mutant also impairs temperature adaptation (1). Together, this information suggests that a balance of UPR induction and resolution is critical for successful temperature adaptation in C. neoformans.The UPR is induced in response to ER stress caused by aberrant protein folding or processing (3-5). A well-conserved feature of UPR activation is the spliceosome-independent removal of an unconventional intron from the mRNA encoding the major UPR transcription factor by the ER-resident transmembrane kinase/ RNase Ire1 (5, 6). In Saccharomyces cerevisiae, this transcription factor is encoded by the HAC1 gene and the unconventional splicing event removes a 252-nucleotide intron from the mRNA, altering the reading frame and allowing production of a functional Hac1p transcription factor. In higher eukaryotes, the orthologous active protein Xbp1 is produced after removal of a 26-nucleotide intron from t...

Puf4 Mediates Post-transcriptional Regulation of Caspofungin Resistance inCryptococcus neoformans

“…Our previous work has implicated the pumilio family RNA binding protein Puf4 in the regulation of ER stress in C. neoformans (27). Puf4 is an effector of calcineurin signaling, a pathway known to regulate thermotolerance and cell integrity (24, 28).…”

“…Puf4 is a positive regulator of the unconventional splicing of the ER-stress transcription factor HXL1 . In contrast, Puf4 is a negative regulator of the ALG7 mRNA, which is stabilized in the puf4Δ mutant (27). Interestingly, the Puf4 elements in the HXL1 mRNA, like FKS1 , are in the 5’ UTR, which may suggest that 5’ UTR Puf4-binding elements exert positive regulatory activity.…”

“…EMSA reactions were set and analyzed as described previously (27). Briefly, all RNA binding reactions contained 5 µg of total protein lysate, 0.5 pmol of the TYE705-labeled oligonucleotide (IDT), and 4 µl 5x EMSA buffer (75 mM HEPES pH 7.4, 200 mM KCl, 25 mM MgCl 2 , 25% glycerol, 5 mM dithiothreitol (DTT), and 0.5 mg/ml yeast tRNA) in a total volume of 20 µl.…”

“…C. neoformans Puf4 is homologous to the Saccharomyces cerevisiae paralogs Puf4 and Mpt5 (26). Our previous work demonstrated that C. neoformans Puf4 recognizes the Mpt5 binding element in its mRNA targets (27). It has been hypothesized that Puf4 may play a role in the regulation of cell wall biosynthesis since the puf4 Δ mutant is resistant to lysing enzymes, is temperature sensitive at both 37°C and 39°C, and is sensitive to Congo red (28).…”

“…Our group has previously shown that Puf4 regulates endoplasmic reticulum (ER)-stress through controlling the splicing of a major ER-stress related transcript, HXL1, and plays a role in the unfolded protein response pathway of C. neoformans. Puf4 also contributes to virulence, as the puf4 Δ mutant has attenuated virulence compared to wild type in an intravenous murine competition model of cryptococcosis (27).…”