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...

Kinetoplastids, including Trypanosoma brucei, control gene expression primarily at the posttranscriptional level. Nuclear mRNA export is an important, but understudied, step in this process. The general heterodimeric export factors, Mex67/Mtr2, function in the export of mRNAs and tRNAs in T. brucei, but RNA binding proteins (RBPs) that regulate export processes by controlling the dynamics of Mex67/Mtr2 ribonucleoprotein formation or transport have not been identified. Here, we report that DRBD18, an essential and abundant T. brucei RBP, associates with Mex67/Mtr2 in vivo, likely through its direct interaction with Mtr2. DRBD18 downregulation results in partial accumulation of poly(A)+ mRNA in the nucleus, but has no effect on localization of intron-containing or mature tRNAs. Comprehensive analysis of transcriptomes from whole cell and cytosol in DRBD18 knockdown parasites demonstrates that depletion of DRBD18 leads to impairment of nuclear export of a subset of mRNAs. CLIP experiments reveal association of DRBD18 with several of these mRNAs. Moreover, DRBD18 knockdown leads to a partial accumulation of the Mex67/Mtr2 export receptors in the nucleus. Taken together, the current study supports a model in which DRBD18 regulates the selective nuclear export of mRNAs by promoting the mobilization of export competent mRNPs to the cytosol through the nuclear pore complex.

Kinetoplastids, including Trypanosoma brucei, control gene expression primarily at the posttranscriptional level. Nuclear mRNA export is an important, but understudied, step in this process. The general heterodimeric export factors, Mex67/Mtr2, function in the export of mRNAs and tRNAs in T. brucei, but RNA binding proteins (RBPs) that regulate export processes by controlling the dynamics of Mex67/Mtr2 ribonucleoprotein formation or transport have not been identified. Here, we report that DRBD18, an essential and abundant T. brucei RBP, associates with Mex67/Mtr2 in vivo, likely through its direct interaction with Mtr2. DRBD18 downregulation results in partial accumulation of poly(A)+ mRNA in the nucleus, but has no effect on the localization of intron‐containing or mature tRNAs. Comprehensive analysis of transcriptomes from whole‐cell and cytosol in DRBD18 knockdown parasites demonstrates that depletion of DRBD18 leads to impairment of nuclear export of a subset of mRNAs. CLIP experiments reveal the association of DRBD18 with several of these mRNAs. Moreover, DRBD18 knockdown leads to a partial accumulation of the Mex67/Mtr2 export receptors in the nucleus. Taken together, the current study supports a model in which DRBD18 regulates the selective nuclear export of mRNAs by promoting the mobilization of export competent mRNPs to the cytosol through the nuclear pore complex.

The basidiomycete fungal pathogen Cryptococcus neoformans requires the PUF protein, Pum1, for hyphal morphogenesis during sexual development. In this study we found that Pum1 was auto-repressive under growth as yeast, but that auto-repression was relieved during filamentous growth through utilization of an alternative transcription start site driven by the master filamentation regulator Znf2. In addition, Pum1 was required to stabilize the ZNF2 mRNA through an indirect mechanism suggesting that Znf2 and Pum1 each positively regulate the expression of the other to achieve the filamentous morphotype required for sexual development in Cryptococcus.

Mating and sexual development in C. neoformans var. grubii strains of the H99 background is often less robust than that laboratory generated isogenic C. neoformans var. neoformans strains in the JEC21 background. In Candida albicans and Saccharomyces serevisiae, slowing of DNA synthesis and engagement of the replication stress response, such as that caused by treatment with hydroxyurea (HU), induces filamentation and pseudohyphal growth, respectively. In this study, we investigated the effect of HU treatment on C. neoformans var. grubii morphogenesis. Treatment with HU did not induce filamentation of yeast cells either in liquid culture or on solid YPD or V8 agar. In the presence of the opposite mating partner, we observed early emergence of hyphae in the presence of HU. Semi-quantitative analysis of fusion using marked strains demonstrated that no significant enhancement of fusion in the presence of HU. Transfer of fusion colonies from crosses performed in the absence of HU to V8 + HU revealed enhanced hyphal growth in the presence of HU. Analysis of expression of the target of HU, ribonucleotide reductase, revealed that a phylogenetically divergent catalytic subunit is replication stress responsive in C. neoformans. These results suggest that induction of replication stress promotes post-fusion hyphal growth of C. neoformans var. grubii strains in the H99 background.

The evolutionarily conserved Pumilio domain-containing RNA binding proteins (RBPs) are involved in many steps of post-transcriptional gene regulation, including RNA stability, polyadenylation, deadenylation, and translation. RBPs are post-translationally modified by methylation of arginine/glycine-rich domains, though the consequences of these modifications are not well known. We determined the arginine methylation and phosphorylation landscape of the Pumilio domain-containing RBP Puf4 from the basidiomycete fungus Cryptococcus neoformans. We found that methyl-deficient Puf4 mutants do not complement critical PUF4 deletion phenotypes, such as resistances to endoplasmic reticulum stress and antifungals, and cell wall remodeling. Methyl-deficient mutants also exhibit unique RNA and protein interactions. Lastly, we identified intra-protein cross talk between post-translationally modified methylated and phosphorylated residues. Overall, we show that post-translational modifications, particularly arginine methylation, of Puf4 regulate the functions of this RBP.

Mating in the human fungal pathogen Cryptococcus neoformans is one mechanism by which spores, the likely infectious propagules are produced. The multistep sexual development process involves mate recognition followed by plasmogamy, filamentation, karyogamy and meiosis ultimately resulting in the basidiospore production. The Puf (Pumilio and FBF) family of RNA binding proteins is known to bind to 3′UTRs of mRNAs and is associated with regulatory functions including translation, stabilization and localization of transcripts. Scanning of 3′UTRs of all the annotated genes of C. neoformans revealed Puf3 binding consensus sequence in the transcripts involved in pheromone signaling cascade. This led us to hypothesize that Puf3 regulates sexual differentiation of C. neoformans. To test this hypothesis we performed bilateral mating assays for wild type and puf3Δ mutants, lacking the Pumilio domain RNA binding protein Puf3. When equal number of opposite mating cells of puf3Δ mutants was cocultured on V8 agar, they were defective in filamentation as compared to the wild type cross. To determine the ability of puf3Δ mutants to produce pheromones and recognize opposite mating partner, northern blot analysis was done. The RNA obtained from mating colonies of puf3Δ mutants bilateral cross was probed for mating pheromone MFα in comparison to the wild type cross (H99α × KN99a ) and the induction of MFα was found to be normal in puf3Δ mutants mating. Our result suggests that mate recognition does occur when puf3Δ mutants are crossed. Future studies will assess the ability of puf3Δ mutants to fuse and undergo post fusion hyphal extension. Verifying the Puf3 binding to mating pheromone signaling transcripts and analyzing the transcriptional level and stability of these transcripts will lead us to define the role of Puf3 in C. neoformans.