Program Book - 27th Fungal Genetics Conference

FULL POSTER SESSION ABSTRACTSconfirmed phosphorylation sites, loss of the individual or neighboring sites have no apparent effect on the free running period length, suggesting that sitesmay work in groups as dynamically regulated charged domains. Some domains promote FRQ stability and lengthen period and other promote turnoverand shorten period. Modifications are dynamic such that at near all times of day “FRQ” describes a heterogeneous mix of proteins with the same aminoacid sequence but variable and distinguishable structure and surface chemistry. We have also used luciferase as a reporter to follow the FRQ-WC coreoscillator under conditions where growth rhythms are manifest. Under these conditions the FRQ/WC oscillator cycles with a normal compensatedcircadian period length even when the overt rhythm of growth moves out of the circadian range.390. Non-optimal codon usage determines the expression level, structure and function of the circadian clock protein FREQUENCY. Mian Zhou 1 , JinhuGuo 5 , Joonseok Cha 1 , Michael Chae 1 , She Chen 2 , Jose Barral 3 , Matthew Sachs 4 , Yi Liu 1 . 1) Department of Physiology, UT Southwestern Medical Center,Dallas, TX; 2) National Institute of Biological Sciences, Beijing, China; 3) Departments of Neuroscience and Cell Biology and Biochemistry and MolecularBiology, The University of Texas Medical Branch, Galveston, TX; 4) Departments of Biology, Texas A&M University, College Station, TX; 5) School of LifeSciences, Sun Yat-sen University, Guangzhou, China.Codon usage bias has been observed in the genomes of almost all organisms and is thought to result from selection for efficient and accurate translationof highly expressed genes 1-3. In addition, codon usage is also implicated in the control of transcription, splicing and RNA structure 4-6. Many genes,however, exhibit little codon usage bias. The lack of codon bias for a gene is thought to be due to lack of selection for mRNA translation. Alternatively,however, non-optimal codon usage may also have biological significance. The rhythmic expression and the proper function of the Neurospora FREQUENCY(FRQ) protein are essential for circadian clock function. Here, we show that, unlike most genes in Neurospora, frq exhibits non-optimal codon usage acrossits entire open reading frame. Optimization of frq codon usage results in the abolition of both overt and molecular circadian rhythms. Codon optimizationnot only increases FRQ expression level but surprisingly, also results in conformational changes in FRQ protein, impaired FRQ phosphorylation, andimpaired functions in the circadian feedback loops. These results indicate that non-optimal codon usage of frq is essential for its circadian clock function.Our study provides an example of how non-optimal codon usage is used to regulate protein expression levels and to achieve optimal protein structure andfunction.391. Two putative long non-coding RNAs upstream of transcription factor Znf2 may regulate morphogenesis (or dimorphic transition) in Cryptococcusneoformans. Nadia Chacko, Linqi Wang, Xiaorong Lin. Biology, Texas A&M University, College Station, TX.Cryptococcus neoformans is an opportunistic human pathogen and the causal agent of fungal meningitis, one of the leading causes of death inimmunocompromised patients. The virulence of this dimorphic fungus is closely tied to its morphology as the yeast form is pathogenic while thefilamentous form is non-pathogenic. The morphological switch from yeast to filament occurs typically during unisexual and bisexual mating but can occurunder mating limiting conditions too. Recently we found that transcription factor Znf2 directs morphological transition from yeast-to-filament and itsactivity is reversely correlated with fungal virulence. The means to increase Znf2 activity, either by activation of its activator or inactivation of itsrepressors, could be of great value to alleviate cryptococcosis. In a search to identify upstream regulators of ZNF2, we screened 60,000 insertional mutantsthat mimic znf2D phenotype. Insertions in two of the selected mutants were found to be in a potential long non-coding RNA located in the intergenicregion of ZNF2. This lncRNA was named RZE1. The rze1D mutant phenotype resembles the znf2D mutant phenotype, supporting our hypothesis that RZE1functions upstream of ZNF2. Interestingly, the expression of RZE1 is increased under host relevant conditions but not under mating-inducing conditions,suggesting that RZE1 could be involved in the adaptation to the host during infection. Surprisingly the expression of ZNF2 is only modestly reduced in theRZE1 insertional mutants, indicating the existence of other potential regulators or non-transcriptional regulation of ZNF2 by RZE1. Further analysis of theintergenic region of ZNF2 revealed the presence of another lncRNA, which was named RZE2. Long ncRNAs are known to regulate genes by transcriptionalactivation, repression and epigenetic control. The investigation of extent of regulatory role of RZE1 and RZE2 on ZNF2, the conditions under which theyexert regulation, and the mode of action (transcriptional, translational, or epigenetic control) of these ncRNAs will further clarify the role of ZNF2 inmorphogenesis and virulence in C. neoformans.392. Introns in Cryptococcus neoformans. Carolin Goebels, Sara Gonzalez-Hilarion, Frédérique Moyrand, Guilhem Janbon. Institut Pasteur, Paris, France.Cryptoccus neoformans is a encapsulated basiomycetous yeast responsible for deadly meningoencephalitis in immunocompromised patients. Theanalysis of its genome sequence revealed that nearly all the genes contain introns. These introns are short (67 bp) and each gene contains 5 introns inaverage. RNAseq data analysis showed that alternative splicing is also very common. Moreover, for most of the genes tested introns appeared to beessential for gene expression. We have studied the pathways by which these introns regulate gene expression in C. neoformans. We indentified a polyAbinding protein as a major key factor in this regulation controlling the degradation of the mRNA transcribed from intronless alleles.393. Unravelling of sexual differentiation mediated by Ire1 via Hxl1-independent manners in Cryptococcus neoformans . Kwang-Woo Jung, Yong-SunBahn. Department of Biotechnology, College of Life science and Biotechnology, Yonsei University, Seoul, South Korea.Sexual differentiation is a key biological process for generating genetically diverse offspring, which contributes to the increased fitness of certain speciesin its environmental niches. A human fungal pathogen, Cryptococcus neoformans, undergoes both bisexual and unisexual differentiations. Our previousstudy revealed that UPR (unfolded protein response) components including an evolutionarily conserved ER stress sensor Ire1 and a unique transcriptionfactor Hxl1 modulate ER stress, cell wall integrity, antifungal drug resistance, and virulence in C. neoformans. In this study, we for the first time provideseveral evidences showing that the UPR pathway governs both bisexual and unisexual differentiation of C. neoformans. In the serotype A strainbackgrounds (H99 and KN99a), the ire1D mutants exhibit severe defects in bilateral cross whereas Hxl1 appears to be dispensable for mating in bothunilateral and bilateral crosses. Cell fusion efficiency of unilateral and bilateral crossing with ire1D mutants is significantly decreased when compared toWT crossing, indicating that Ire1 promotes cell-to-cell fusion during mating. Moreover, deletion of the IRE1 gene blocked induction of pheromonemediatedconjugation tubes in crg1D mutants, which lack a RGS protein that negatively regulates pheromone responsive G-protein signaling.Unexpectedly, however, expression of the mating pheromone gene (MFa1) was strongly induced by the mutation of IRE1 gene in serotype A strain,suggesting that Ire1 has both positive and negative roles in mating of serotype A C. neoformans. The ire1D mutants constructed in serotype D (JEC21a andJEC20a) strains also exhibit mating defects similar serotype A ire1D mutant strains, whereas the hxl1D mutants are dispensable for mating. It indicates thatthe role of Ire1 in sexual differentiation is evolutionary conserved in both serotype A and D strains. The unisexual mating, also known as monokaryoticfruiting, is an alternative differentiation process in C. neoformans. The deletion of IRE1 in the XL280 strain, which is used as a tester strain for same-sexmating, causes significant defects in filamentation whereas the hxl1D mutants exhibit levels of filamentation indistinguishable from those of XL280. Inconclusion, the Ire1 regulates both bisexual and unisexual mating of C. neoformans in Hxl1-independent manners.216