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

Star Formation History of a Young Super-Star Cluster in NGC 4038/39: Direct Detection of Low Mass Pre-Main Sequence Stars

We present an analysis of the near-infrared spectrum of a young massive star cluster in the overlap region of the interacting galaxies NGC 4038/39 using population synthesis models. Our goal is to model the cluster population as well as provide rough constraints on its initial mass function (IMF). The cluster shows signs of youth such as thermal radio emission and strong hydrogen emission lines in the near-infrared. Late-type absorption lines are also present which are indicative of late-type stars in the cluster. The strength and ratio of these absorption lines cannot be reproduced through either late-type pre-main sequence (PMS) stars or red supergiants alone. Thus we interpret the spectrum as a superposition of two star clusters of different ages, which is feasible since the 1" spectrum encompasses a physical region of ~90 pc and radii of super-star clusters are generally measured to be a few parsecs. One cluster is young (<= 3 Myr) and is responsible for part of the late-type absorption features, which are due to PMS stars in the cluster, and the hydrogen emission lines. The second cluster is older (6 Myr - 18 Myr) and is needed to reproduce the overall depth of the late-type absorption features in the spectrum. Both are required to accurately reproduce the near-infrared spectrum of the object. Thus we have directly detected PMS objects in an unresolved super-star cluster for the first time using a combination of population synthesis models and pre-main sequence tracks. This analysis serves as a testbed of our technique to constrain the low-mass IMF in young super-star clusters as well as an exploration of the star formation history of young UC HII regions.Comment: 26 pages, 5 figures, accepted for publication in the Astrophysical Journa

Force weighting approach to calculate spinal cumulative loading for ergonomic workforce planning in production

For the prevention of musculoskeletal disorders (MSD), the evaluation of manual materials handling (MMH) is important. In this context, cumulative loading can be used as an exposure index for the ergonomic assessment of workplaces. However, it was shown in previous empirical studies that most existing methods for calculating cumulative loading fail to completely capture the resulting physiological effects of working conditions on human workers. Therefore, this contribution outlines the development and validation of a novel force weighted approach to calculated spinal cumulative loading that reflects the muscular exposure. Empirical data from 36 individuals were used as the data basis for deriving and validating the calculation method. The results of the validation show a high prediction quality on the basis of the hold-out method. Hence, the method provides relevant indicators for the ergonomic assessment of MMH activities. Thus, it might be a useful tool for workforce planning in production

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

The Antennae Galaxies (NGC 4038/4039) Revisited: ACS and NICMOS Observations of a Prototypical Merger

The ACS and NICMOS have been used to obtain new HST images of NGC 4038/4039 ("The Antennae"). These new observations allow us to better differentiate compact star clusters from individual stars, based on both size and color. We use this ability to extend the cluster luminosity function by approximately two magnitudes over our previous WFPC2 results, and find that it continues as a single power law, dN/dL propto L^alpha with alpha=-2.13+/-0.07, down to the observational limit of Mv~-7. Similarly, the mass function is a single power law dN/dM propto M^beta with beta=-2.10+/-0.20 for clusters with ages t<3x10^8 yr, corresponding to lower mass limits that range from 10^4 to 10^5 Msun, depending on the age range of the subsample. Hence the power law indices for the luminosity and mass functions are essentially the same. The luminosity function for intermediate-age clusters (i.e., ~100-300 Myr old objects found in the loops, tails, and outer areas) shows no bend or turnover down to Mv~-6, consistent with relaxation-driven cluster disruption models which predict the turnover should not be observed until Mv~-4. An analysis of individual ~0.5-kpc sized areas over diverse environments shows good agreement between values of alpha and beta, similar to the results for the total population of clusters in the system. Several of the areas studied show evidence for age gradients, with somewhat older clusters appearing to have triggered the formation of younger clusters. The area around Knot B is a particularly interesting example, with an ~10-50 Myr old cluster of estimated mass ~10^6 Msun having apparently triggered the formation of several younger, more massive (up to 5x10^6 Msun) clusters along a dust lane.Comment: 84 pages, 9 tables, 31 figures; ApJ accepte