Program Book - 27th Fungal Genetics Conference

CONCURRENT SESSION ABSTRACTSquantifying their usage under different nutritional conditions (rich and minimal media, carbon and nitrogen starvation) in the wild type and the Drbp35mutant. Results of these polyadenylation maps will be presented, including candidate APA targets, sequence motifs present in long 3' UTRs, Rbp35-dependent mRNA isoforms, and conservation of significant mRNA isoforms in other filamentous fungi.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.Dual targeting of glycolytic enzymes by alternative splicing and translational read-through. Johannes Freitag, Julia Ast, Alina Stiebler, Michael Bölker.Department of Biology, Philipps-Universität Marburg, Marburg, Germany.Processing of mRNA is a highly conserved process in eukaryotes involving three major steps. Nascent transcripts are capped at their 5’end, introns areremoved by splicing and the 3’end is cleaved and polyadenylated. In the plant pathogenic fungus Ustilago maydis, several genes show hallmarks ofdifferential splicing and alternative polyadenylation resulting in the production of C-terminally extended proteins. We detected that this process leads togeneration of an extended glyceraldehyde-3-phosphate dehydrogenase (GAPDH) isoform harboring a C-terminal peroxisomal targeting sequence (PTS1).We could also detect peroxisomal isoforms of two further glycolytic enzymes, phosphoglycerate kinase (PGK) and triosephosphate isomerase (TPI).Remarkably, peroxisomal isoforms of PGK and TPI are generated by translational read-through in U. maydis. Further analysis revealed that dual targetingof glycolytic enzymes to peroxisomes and the cytoplasm is not restricted to U. maydis but occurs in a variety of fungal species. Interestingly, in differentspecies variable mechanisms to generate extended peroxisomal isoforms of glycolytic enzymes are operating. In the ascomycete Aspergillus nidulans thePTS1-motif of PGK is derived from alternative splicing and polyadenylation, while translational read-through is used to generate a peroxisomal isoform ofGAPDH. We could also show that some enzymes are partially targeted to peroxisomes by means of weak peroxisomal targeting signals. Dual localization ofglycolytic enzymes to peroxisomes and the cytoplasm appears to be widespread in fungi. This indicates that fungal peroxisomes are endowed with a morecomplex metabolism than previously assumed. Thus, the consideration of alternative splicing and translational read-through will be of importance infuture proteomic and metabolomic studies of organelles.Non-optimal codon usage determines the expression level, structure and function of the circadian clock protein FREQUENCY. Mian Zhou 1 , Jinhu Guo 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 Molecular Biology, TheUniversity of Texas Medical Branch, Galveston, TX; 4) Departments of Biology, Texas A&M University, College Station, TX; 5) School of Life Sciences, SunYat-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.A transcriptome-wide view on microtubule-dependent mRNA transport. Carl Haag 1 , Julian Konig 2 , Kathi Zarnack 3 , Michael Feldbrugge 1 . 1) Institut forMicrobiology, Heinrich-Heine University, Düsseldorf, NRW, Germany; 2) MRC LMB Cambridge, UK; 3) EBI Hinxton, UK.Long distance transport of mRNAs regulates spatio-temporal gene expression during polar growth. In filaments of U. maydis, for example, microtubuledependentshuttling of mRNAs is crucial to determine the axis of polarity. The key component of this transport system is the RNA-binding protein Rrm4that binds a distinct set of target mRNAs. Recently, we discovered a novel mechanism for mRNA transport, namely the co-transport of Rrm4 andassociated mRNAs with endosomes. Here, new insights on mRNA transport will be presented using the improved in vivo UV-crosslinking technique: iCLIP.This technique allows identification of target mRNAs at the transcriptome-wide level with single nucleotide resolution.54