Program Book - 27th Fungal Genetics Conference

FULL POSTER SESSION ABSTRACTS394. Functional analysis of PUF mediated post-transcriptional regulation in Cryptococcus neoformans. Jan Naseer Kaur, John Panepinto. University atBuffalo, Buffalo, NY.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 includingtranslation, stabilization and localization of transcripts. Investigation of the C. neoformans genome has revealed that it encodes four PUF proteins. PUFproteins are typically characterized by the presence of 8 consecutive Puf repeats, however variations do occur. Sequence analysis and evolutionary studiesof Puf proteins in fungi has predicted Puf1, Puf2 and Puf3 of C. neoformans to contain 5, 3 and 8 PUM-HD repeats respectively. We hypothesize that Puf1,Puf2 and Puf3 act as RBPs and regulate gene expression. The PUF proteins characterized to date have been reported to bind to 3’ UTR sequenceencompassing a UGUR tetranucleotide in their target RNA. Previous studies have reported that the core motif of UGUA followed by a variable region thatPuf3 binds to is conserved from yeast to humans. Scanning of 3’UTRs of all the annotated genes of C. neoformans revealed Puf3 binding consensussequence in the transcripts involved in pheromone signaling cascade. To test this hypothesis we performed bilateral mating assays for wild type and puf3Dmutants. When equal numbers of opposite mating cells of puf3D mutants were cocultured on V8 agar, we observed that they were defective infilamentation as compared to the wild type cross. To determine the ability of puf3D mutants to produce pheromone, northern blot was done. The RNAobtained from puf3D mutants bilateral cross was probed for mating pheromone MFa in comparison to the wild type cross (H99a x KN99a) and theinduction of MFa was found to be normal in puf3D mutants mating. Also, fusant colony formation assay revealed that filamention defect of puf3D is notdue to impaired cell fusion. Using fluorescent microscopy we have shown that mCherry tagged Puf3 localizes to areas of hyphal growth. Our resultssuggest that mate recognition and fusion do occur when puf3D mutants are crossed. We predict that the defect is in Puf3 mediated post fusion hyphalextension. Future studies will determine the mechanism of Puf3 regulation on potential target transcripts. Also we will identify the target/s of Puf1 andPuf2 and their mechanism of regulation which would enable us to establish a link between the physiology and the Puf regulon of C. neoformans.395. Determining the direct targets of two master regulators of sexual development in Cryptococcus neoformans. Matthew E Mead 1 , Emilia K Kruzel 1 ,Christina M Hull 1,2 . 1) Department of Biomolecular Chemistry, University of Wisconsin School of Medicine and Public Health, Madison, WI, USA, 53706; 2)Department of Medical Microbiology and Immunology, University of Wisconsin School of Medicine and Public Health, Madison, WI, USA, 53706.Cryptococcus neoformans is a major global fungal pathogen that causes disease primarily in immunocompromised individuals. Proposed infectiousparticles include spores, which are produced as a result of sexual development. In crosses between a and a cells this process is in part controlled by thehomeodomain transcription factors Sxi1a and Sxi2a. To understand the molecular events governing development, we set out to identify directtranscriptional targets of the Sxi1a/Sxi2a heterodimer.First, we created a haploid strain in which galactose-inducible promoters control the expression of SXI1a and SXI2a. This SXI-inducible strain allowed usto assess global transcript levels in the presence and absence of SXI expression. At the same time, we compared changes in transcript levels in crossesbetween wild type (a x a) and sxi deletion (sxi1aD x sxi2aD) strains. We discovered that 185 genes exhibited a “Sxi-induced” regulation pattern in bothexperiments.Upstream regions of these highly regulated genes were then analyzed using motif-finding algorithms, and a subset of the Sxi-induced genes was found tocontain a sequence similar to one bound by Sxi1a/Sxi2a in vitro. Individual occurrences of the motif were tested in a Yeast 1-Hybrid system and shown tobe bound by Sxi1a/Sxi2a in a sequence-specific and heterodimer-specific manner. An in vivo reporter assay was then used to show that these binding sitesconfer Sxi-dependent regulation to their downstream targets that is also sequence specific.The list of direct targets studied so far includes numerous uncharacterized genes and putative transcriptional regulators likely important for controllingsubsequent developmental transitions. Future studies will focus on building a complete, Sxi-dependent transcriptional network of development. This workwill help us better understand a process that results in the production of a likely infectious particle in mammalian disease.MEM is funded by the Microbes in Health and Disease Training Grant (NIH T32 AI55397).396. Post-transcriptional gene regulation contributes to host temperature adaptation and virulence in Cryptococcus neoformans. Amanda L. MisenerBloom 1,2 , Kurtis Downey 1 , Nathan K. Wool 1 , John C. Panepinto 1,2 . 1) Microbiology/Immunology, SUNY University at Buffalo, Buffalo, NY; 2) Witebsky Centerfor Microbial Pathogenesis and Immunology, SUNY University at Buffalo, Buffalo, NY.In response to the hostile host environment, pathogens must undergo rapid reprogramming of gene expression to adapt to the stresses they encounter.Upon exposure to host temperature, Ribosomal protein (RP) transcripts are rapidly repressed in C. neoformans. We are interested in investigating specificmechanisms involved in this response, as this repression may be a critical process in host temperature adaptation. Using a mutant null of the majordeadenylase, Ccr4, we have discovered that this repression is in part due to enhanced degradation of RP-transcripts. Ccr4 lacks a nucleic acid bindingdomain and therefore must be recruited to mRNA targets via RNA binding proteins. Using MEME analysis and chromatographic techniques, we haveidentified a shared cis element in the 3’UTR of RP transcripts that is recognized by the zinc knuckle protein, Gis2. We are currently investigating theimportance of this protein-RNA interaction in the expression of RP genes.Host temperature-induced enhanced degradation of RP transcripts is also dependent on the dissociable RNA polymerase II subunit, Rpb4. Specifically, wedemonstrated that in an rpb4D mutant, RP-transcript deadenylation is impaired, suggesting that Rpb4 may be required for Ccr4-targeted degradation. Inaddition, we observed that upon a shift to 37°C, Rpb4 travels from the nucleus to the cytoplasm, supporting a role for Rpb4 in coupling transcription anddegradation. Interestingly, this coupling is not restricted to the RP transcripts, as Rpb4 is also involved in enhanced decay of ER stress transcripts followingtheir peak induction, one hour after a shift to host temperature. We have demonstrated that signaling through PKH enhances the degradation of the RPtranscriptsin response to host temperature, but not the ER stress transcripts, highlighting the complexity of this system. We report that whentranscription and degradation are uncoupled by the loss of Rpb4, growth at host temperature is impaired and virulence in a mouse model of disseminatedcryptococcosis is attenuated. Our data suggests that coupling of transcription and degradation via Rpb4 allows the cell to control the intensity andduration of different responses at specific times following exposure to host temperature, contributing to the ability of C. neoformans to adapt to thisstress.397. Protein arginine methylation in post-transcriptional gene regulation and stress adaptation of Cryptococcus neoformans. J.T. Graham Solomons,Amanda L.M. Bloom, John C. Panepinto. The Department of Microbiology and Immunology, The State University of New York at Buffalo, Buffalo, NY.Cryptococcus neoformans is environmental fungus that opportunistically infects immune compromised individuals, and is widely studied as a modelbasidiomycete. The ability to adapt to host temperature is an essential pathogenic trait of C. neoformans, and the degradation of mRNA initiated by themajor deadenylase Ccr4 appears to play an integral role in the temperature stress response of C. neoformans. Microarray analysis revealed that the largestfunctionally related group of mRNA stabilized in the ccr4D mutant encode ribosomal protein (RP) transcripts. An RNA-binding protein, identified as Gis2,has been shown to interact specifically with a cis element in the 3’UTR of RP transcripts. Gis2 is a small protein (19kDa), predominantly comprised of a27th Fungal Genetics Conference | 217