EXPLORING FATC DOMAIN FUNCTION IN YEAST TRA1

Tral is an essential yeast protein required for regulated transcription. Its human homolog TRRAP regulates factors important in oncogenesis. Mutation of leucine to alanine at position 3733 in the FATC domain {tralla) results in growth phenotypes including sensitivity to ethanol. My aim was to examine genetic interactions o f the FA TC domain o f Tral to define its cellular role. I screened for extragenic suppressors of the ethanol sensitivity caused by tralla, identifying an opal mutation at tryptophan 165 of NAM7 as a suppressor. Deleting nam7, upf3, or nmd2 similarly suppressed tralLA, thereby linking Tral to nonsense mediated decay. I propose that Tral regulates transcription of genes also regulated by NMD. This work emphasizes the importance of NMD in gene regulation. Furthermore, the cross regulation between Tral and NMD suggests that mutations in the human NMD machinery may provide a mechanism to alter pathways influenced by TRRAP in human disease

Loss of nonsense mediated decay suppresses mutations in Saccharomyces cerevisiae TRA1

<p>Abstract</p> <p>Background</p> <p>Tra1 is an essential protein in <it>Saccharomyces cerevisiae</it>. It was first identified in the SAGA and NuA4 complexes, both with functions in multiple aspects of gene regulation and DNA repair, and recently found in the ASTRA complex. Tra1 belongs to the PIKK family of proteins with a C-terminal PI3K domain followed by a FATC domain. Previously we found that mutation of leucine to alanine at position 3733 in the FATC domain of Tra1 (<it>tra1-L3733A</it>) results in transcriptional changes and slow growth under conditions of stress. To further define the regulatory interactions of Tra1 we isolated extragenic suppressors of the <it>tra1-L3733A </it>allele.</p> <p>Results</p> <p>We screened for suppressors of the ethanol sensitivity caused by <it>tra1-L3733A</it>. Eleven extragenic recessive mutations, belonging to three complementation groups, were identified that partially suppressed a subset of the phenotypes caused by tra<it>1-L3733A</it>. Using whole genome sequencing we identified one of the mutations as an opal mutation at tryptophan 165 of <it>UPF1/NAM7</it>. Partial suppression of the transcriptional defect resulting from <it>tra1-L3733A </it>was observed at <it>GAL10</it>, but not at <it>PHO5</it>. Suppression was due to loss of nonsense mediated decay (NMD) since deletion of any one of the three NMD surveillance components (<it>upf1/nam7, upf2/nmd2</it>, or <it>upf3</it>) mediated the effect. Deletion of <it>upf1 </it>suppressed a second FATC domain mutation, <it>tra1-F3744A</it>, as well as a mutation to the PIK3 domain. In contrast, deletions of SAGA or NuA4 components were not suppressed.</p> <p>Conclusions</p> <p>We have demonstrated a genetic interaction between <it>TRA1 </it>and genes of the NMD pathway. The suppression is specific for mutations in <it>TRA1</it>. Since NMD and Tra1 generally act reciprocally to control gene expression, and the FATC domain mutations do not directly affect NMD, we suggest that suppression occurs as the result of overlap and/or crosstalk in these two broad regulatory networks.</p

Mutational analysis of the C-terminal FATC domain of Saccharomyces cerevisiae Tra1

Tra1 is a component of the Saccharomyces cerevisiae SAGA and NuA4 complexes and a member of the PIKK family, which contain a C-terminal phosphatidylinositol 3-kinase-like (PI3K) domain followed by a 35-residue FATC domain. Single residue changes of L3733A and F3744A, within the FATC domain, resulted in transcriptional changes and phenotypes that were similar but not identical to those caused by mutations in the PI3K domain or deletions of other SAGA or NuA4 components. The distinct nature of the FATC mutations was also apparent from the additive effect of tra1-L3733A with SAGA, NuA4, and tra1 PI3K domain mutations. Tra1-L3733A associates with SAGA and NuA4 components and with the Gal4 activation domain, to the same extent as wild-type Tra1; however, steady-state levels of Tra1-L3733A were reduced. We suggest that decreased stability of Tra1-L3733A accounts for the phenotypes since intragenic suppressors of tra1-L3733A restored Tra1 levels, and reducing wild-type Tra1 led to comparable growth defects. Also supporting a key role for the FATC domain in the structure/function of Tra1, addition of a C-terminal glycine residue resulted in decreased association with Spt7 and Esa1, and loss of cellular viability. These findings demonstrate the regulatory potential of mechanisms targeting the FATC domains of PIKK proteins

First fungal genome sequence from Africa : a preliminary analysis

Some of the most significant breakthroughs in the biological sciences this century will emerge from the development of next generation sequencing technologies. The ease of availability of DNA sequence made possible through these new technologies has given researchers opportunities to study organisms in a manner that was not possible with Sanger sequencing. Scientists will, therefore, need to embrace genomics, as well as develop and nurture the human capacity to sequence genomes and utilise the ’tsunami‘ of data that emerge from genome sequencing. In response to these challenges, we sequenced the genome of Fusarium circinatum, a fungal pathogen of pine that causes pitch canker, a disease of great concern to the South African forestry industry. The sequencing work was conducted in South Africa, making F. circinatum the first eukaryotic organism for which the complete genome has been sequenced locally. Here we report on the process that was followed to sequence, assemble and perform a preliminary characterisation of the genome. Furthermore, details of the computer annotation and manual curation of this genome are presented. The F. circinatum genome was found to be nearly 44 million bases in size, which is similar to that of four other Fusarium genomes that have been sequenced elsewhere. The genome contains just over 15 000 open reading frames, which is less than that of the related species, Fusarium oxysporum, but more than that for Fusarium verticillioides. Amongst the various putative gene clusters identified in F. circinatum, those encoding the secondary metabolites fumosin and fusarin appeared to harbour evidence of gene translocation. It is anticipated that similar comparisons of other loci will provide insights into the genetic basis for pathogenicity of the pitch canker pathogen. Perhaps more importantly, this project has engaged a relatively large group of scientists including students in a significant genome project that is certain to provide a platform for growth in this important area of research in the future.We thank the National Research Foundation (NRF) of South Africa, members of the Tree Protection Co-operative Programme, the THRIP initiative of the Department of Trade and Industry and the Department of Science and Technology (DST)/NRF Centre of Excellence in Tree Health Biotechnology and the Oppenheimer Foundation for funding.http://www.sajs.co.zanf201