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

Requirements for E1A dependent transcription in the yeast Saccharomyces cerevisiae

<p>Abstract</p> <p>Background</p> <p>The human adenovirus type 5 early region 1A (E1A) gene encodes proteins that are potent regulators of transcription. E1A does not bind DNA directly, but is recruited to target promoters by the interaction with sequence specific DNA binding proteins. In mammalian systems, E1A has been shown to contain two regions that can independently induce transcription when fused to a heterologous DNA binding domain. When expressed in <it>Saccharomyces cerevisiae</it>, each of these regions of E1A also acts as a strong transcriptional activator. This allows yeast to be used as a model system to study mechanisms by which E1A stimulates transcription.</p> <p>Results</p> <p>Using 81 mutant yeast strains, we have evaluated the effect of deleting components of the ADA, COMPASS, CSR, INO80, ISW1, NuA3, NuA4, Mediator, PAF, RSC, SAGA, SAS, SLIK, SWI/SNF and SWR1 transcriptional regulatory complexes on E1A dependent transcription. In addition, we examined the role of histone H2B ubiquitylation by Rad6/Bre1 on transcriptional activation.</p> <p>Conclusion</p> <p>Our analysis indicates that the two activation domains of E1A function via distinct mechanisms, identify new factors regulating E1A dependent transcription and suggest that yeast can serve as a valid model system for at least some aspects of E1A function.</p

Systematic genetic array analysis links the Saccharomyces cerevisiae SAGA/SLIK and NuA4 component Tra1 to multiple cellular processes

<p>Abstract</p> <p>Background</p> <p>Tra1 is an essential 437-kDa component of the <it>Saccharomyces cerevisiae </it>SAGA/SLIK and NuA4 histone acetyltransferase complexes. It is a member of a group of key signaling molecules that share a carboxyl-terminal domain related to phosphatidylinositol-3-kinase but unlike many family members, it lacks kinase activity. To identify genetic interactions for <it>TRA1 </it>and provide insight into its function we have performed a systematic genetic array analysis (SGA) on <it>tra1</it>_{<it>SRR</it>3413}, an allele that is defective in transcriptional regulation.</p> <p>Results</p> <p>The SGA analysis revealed 114 synthetic slow growth/lethal (SSL) interactions for <it>tra1</it>_{<it>SRR</it>3413}. The interacting genes are involved in a range of cellular processes including gene expression, mitochondrial function, and membrane sorting/protein trafficking. In addition many of the genes have roles in the cellular response to stress. A hierarchal cluster analysis revealed that the pattern of SSL interactions for <it>tra1</it>_{<it>SRR</it>3413}most closely resembles deletions of a group of regulatory GTPases required for membrane sorting/protein trafficking. Consistent with a role for Tra1 in cellular stress, the <it>tra1</it>_{<it>SRR</it>3413}strain was sensitive to rapamycin. In addition, calcofluor white sensitivity of the strain was enhanced by the protein kinase inhibitor staurosporine, a phenotype shared with the Ada components of the SAGA/SLIK complex. Through analysis of a GFP-Tra1 fusion we show that Tra1 is principally localized to the nucleus.</p> <p>Conclusion</p> <p>We have demonstrated a genetic association of Tra1 with nuclear, mitochondrial and membrane processes. The identity of the SSL genes also connects Tra1 with cellular stress, a result confirmed by the sensitivity of the <it>tra1</it>_{<it>SRR</it>3413}strain to a variety of stress conditions. Based upon the nuclear localization of GFP-Tra1 and the finding that deletion of the Ada components of the SAGA complex result in similar phenotypes as <it>tra1</it>_{<it>SRR</it>3413}, we suggest that the effects of <it>tra1</it>_{<it>SRR</it>3413}are mediated, at least in part, through its role in the SAGA complex.</p

C-terminal processing of yeast Spt7 occurs in the absence of functional SAGA complex

<p>Abstract</p> <p>Background</p> <p>Spt7 is an integral component of the multi-subunit SAGA complex that is required for the expression of ~10% of yeast genes. Two forms of Spt7 have been identified, the second of which is truncated at its C-terminus and found in the SAGA-like (SLIK) complex.</p> <p>Results</p> <p>We have found that C-terminal processing of Spt7 to its SLIK form (Spt7_SLIK) and to a distinct third form (Spt7_Form3) occurs in the absence of the SAGA complex components Gcn5, Spt8, Ada1 and Spt20, the latter two of which are required for the integrity of the complex. In addition, N-terminally truncated derivatives of Spt7, including a derivative lacking the histone fold, are processed, indicating that the C-terminus of Spt7 is sufficient for processing and that processing does not require functional Spt7. Using galactose inducible Spt7 expression, we show that the three forms of Spt7 appear and disappear at approximately the same rate with full-length Spt7 not being chased into Spt7_SLIKor Spt7_Form3. Interestingly, reduced levels of Spt7_SLIKand Spt7_Form3were observed in a strain lacking the SAGA component Ubp8, suggesting a regulatory role for Ubp8 in the truncation of Spt7.</p> <p>Conclusion</p> <p>We conclude that truncation of Spt7 occurs early in the biosynthesis of distinct Spt7 containing complexes rather than being a dynamic process linked to the action of the SAGA complex in transcriptional regulation.</p

A novel mistranslating tRNA model in Drosophila melanogaster has diverse, sexually dimorphic effects

Transfer RNAs (tRNAs) are the adaptor molecules required for reading the genetic code and producing proteins. Transfer RNA variants can lead to genome-wide mistranslation, the misincorporation of amino acids not specified by the standard genetic code into nascent proteins. While genome sequencing has identified putative mistranslating transfer RNA variants in human populations, little is known regarding how mistranslation affects multicellular organisms. Here, we create a multicellular model of mistranslation by integrating a serine transfer RNA variant that mistranslates serine for proline (tRNAUGG,G26ASer) into the Drosophila melanogaster genome. We confirm mistranslation via mass spectrometry and find that tRNAUGG,G26ASer misincorporates serine for proline at a frequency of ∼0.6% per codon. tRNAUGG,G26ASer extends development time and decreases the number of flies that reach adulthood. While both sexes of adult flies containing tRNAUGG,G26ASer present with morphological deformities and poor climbing performance, these effects are more pronounced in female flies and the impact on climbing performance is exacerbated by age. This model will enable studies into the synergistic effects of mistranslating transfer RNA variants and disease-causing alleles

SSGSS: The Spitzer-SDSS-GALEX Spectroscopic Survey

The Spitzer-SDSS-GALEX Spectroscopic Survey (SSGSS) provides a new sample of 101 star-forming galaxies at z < 0.2 with unprecedented multi-wavelength coverage. New mid- to far-infrared spectroscopy from the Spitzer Space Telescope is added to a rich suite of previous imaging and spectroscopy, including ROSAT, Galaxy Evolution Explorer, Sloan Digital Sky Survey, Two Micron All Sky Survey, and Spitzer/SWIRE. Sample selection ensures an even coverage of the full range of normal galaxy properties, spanning two orders of magnitude in stellar mass, color, and dust attenuation. In this paper we present the SSGSS data set, describe the science drivers, and detail the sample selection, observations, data reduction, and quality assessment. Also in this paper, we compare the shape of the thermal continuum and the degree of silicate absorption of these typical, star-forming galaxies to those of starburst galaxies. We investigate the link between star formation rate, infrared luminosity, and total polycyclic aromatic hydrocarbon luminosity, with a view to calibrating the latter for spectral energy distribution models in photometric samples and at high redshift. Last, we take advantage of the 5-40 micron spectroscopic and far-infrared photometric coverage of this sample to perform detailed fitting of the Draine et al. dust models, and investigate the link between dust mass and star formation history and active galactic nucleus properties.Comment: 60 pages, 20 figure

Mid-Infrared Spectral Measures of Star-Formation and AGN Activity in Normal Galaxies

We investigate the use of MIR PAH bands, continuum and emission lines as probes of star-formation and AGN activity in a sample of 100 'normal' and local (z~0.1) emission-line galaxies. The MIR spectra were obtained with the Spitzer Space Telescope Infrared Spectrograph (IRS) as part of the Spitzer-SDSS-GALEX Spectroscopic Survey (SSGSS) which includes multi-wavelength photometry from the UV to the FIR and optical spectroscopy. The continuum and features were extracted using PAHFIT (Smith et al. 2007), a decomposition code which we find to yield PAH equivalent widths up to ~30 times larger than the commonly used spline methods. Despite the lack of extreme objects in our sample (such as strong AGNs, low metallicity galaxies or ULIRGs), we find significant variations in PAH, continuum and emission line properties and systematic trends between these MIR properties and optically derived physical properties such as age, metallicity and radiation field hardness. We revisit the diagnostic diagram relating PAH equivalent widths and [Ne II]12.8micrometers/[O IV]25.9micrometers line ratios and find it to be in much better agreement with the standard optical star-formation/AGN classification than when spline decompositions are used, while also potentially revealing obscured AGNs. The luminosity of individual PAH components, of the continuum, and with poorer statistics, of the neon emission lines and molecular hydrogen lines, are found to be tightly correlated to the total IR luminosity, making individual MIR components good gauges of the total dust emission in SF galaxies. Like the total IR luminosity, these individual components can be used to estimate dust attenuation in the UV and in Halpha lines based on energy balance arguments. We also propose average scaling relations between these components and dust corrected, Halpha derived star-formation rates.Comment: Accepted for publication in Ap

Optimization of growth media components for polyhydroxyalkanoate (PHA) production from organic acids by Ralstonia eutropha

We employed systematic mixture analysis to determine optimal levels of acetate, propionate, and butyrate for cell growth and polyhydroxyalkanoate (PHA) production by Ralstonia eutropha H16. Butyrate was the preferred acid for robust cell growth and high PHA production. The 3-hydroxyvalerate content in the resulting PHA depended on the proportion of propionate initially present in the growth medium. The proportion of acetate dramatically affected the final pH of the growth medium. A model was constructed using our data that predicts the effects of these acids, individually and in combination, on cell dry weight (CDW), PHA content (%CDW), PHA production, 3HV in the polymer, and final culture pH. Cell growth and PHA production improved approximately 1.5-fold over initial conditions when the proportion of butyrate was increased. Optimization of the phosphate buffer content in medium containing higher amounts of butyrate improved cell growth and PHA production more than 4-fold. The validated organic acid mixture analysis model can be used to optimize R. eutropha culture conditions, in order to meet targets for PHA production and/or polymer HV content. By modifying the growth medium made from treated industrial waste, such as palm oil mill effluent, more PHA can be produced.Malaysia. Ministry of Science, Technology and Innovation (MOSTI

A chemical survey of exoplanets with ARIEL

Thousands of exoplanets have now been discovered with a huge range of masses, sizes and orbits: from rocky Earth-like planets to large gas giants grazing the surface of their host star. However, the essential nature of these exoplanets remains largely mysterious: there is no known, discernible pattern linking the presence, size, or orbital parameters of a planet to the nature of its parent star. We have little idea whether the chemistry of a planet is linked to its formation environment, or whether the type of host star drives the physics and chemistry of the planet’s birth, and evolution. ARIEL was conceived to observe a large number (~1000) of transiting planets for statistical understanding, including gas giants, Neptunes, super-Earths and Earth-size planets around a range of host star types using transit spectroscopy in the 1.25–7.8 μm spectral range and multiple narrow-band photometry in the optical. ARIEL will focus on warm and hot planets to take advantage of their well-mixed atmospheres which should show minimal condensation and sequestration of high-Z materials compared to their colder Solar System siblings. Said warm and hot atmospheres are expected to be more representative of the planetary bulk composition. Observations of these warm/hot exoplanets, and in particular of their elemental composition (especially C, O, N, S, Si), will allow the understanding of the early stages of planetary and atmospheric formation during the nebular phase and the following few million years. ARIEL will thus provide a representative picture of the chemical nature of the exoplanets and relate this directly to the type and chemical environment of the host star. ARIEL is designed as a dedicated survey mission for combined-light spectroscopy, capable of observing a large and well-defined planet sample within its 4-year mission lifetime. Transit, eclipse and phase-curve spectroscopy methods, whereby the signal from the star and planet are differentiated using knowledge of the planetary ephemerides, allow us to measure atmospheric signals from the planet at levels of 10–100 part per million (ppm) relative to the star and, given the bright nature of targets, also allows more sophisticated techniques, such as eclipse mapping, to give a deeper insight into the nature of the atmosphere. These types of observations require a stable payload and satellite platform with broad, instantaneous wavelength coverage to detect many molecular species, probe the thermal structure, identify clouds and monitor the stellar activity. The wavelength range proposed covers all the expected major atmospheric gases from e.g. H2O, CO2, CH4 NH3, HCN, H2S through to the more exotic metallic compounds, such as TiO, VO, and condensed species. Simulations of ARIEL performance in conducting exoplanet surveys have been performed – using conservative estimates of mission performance and a full model of all significant noise sources in the measurement – using a list of potential ARIEL targets that incorporates the latest available exoplanet statistics. The conclusion at the end of the Phase A study, is that ARIEL – in line with the stated mission objectives – will be able to observe about 1000 exoplanets depending on the details of the adopted survey strategy, thus confirming the feasibility of the main science objectives.Peer reviewedFinal Published versio