Project description:Alterations in global mRNA decay can broadly impact multiple upstream and downstream stages of gene expression. For example, accelerated cytoplasmic mRNA degradation can trigger a reduction in mammalian RNA polymerase II (RNAPII) transcription, although signals that connect these seemingly distal processes remain largely unknown. Here, we used tandem mass tag labeling with mass spectrometry to chart how changes in Xrn1-dependent mRNA degradation impact nuclear-cytoplasmic protein distribution in human cells. Notably, accelerating mRNA decay through expression of a gammaherpesviral endonuclease known to coordinate with Xrn1 drove nuclear relocalization of many RNA binding proteins. Particularly enriched in the relocalized subset were factors linked to the poly(A) tail. Conversely, cells lacking Xrn1 exhibited changes in the localization and/or abundance of numerous factors linked to mRNA turnover. Based on these data, we uncovered a new role for cytoplasmic poly(A) binding protein in repressing RNAPII transcription upon its mRNA decay-induced translocation to the nucleus.
Project description:We explored genomic expression patterns in the yeast Saccharomyces cerevisiae responding to diverse environmental transitions. DNA microarrays were used to measure changes in transcript levels over time for almost every yeast gene, as cells responded to temperature shocks, hydrogen peroxide, the superoxide-generating drug menadione, the sulfhydryl-oxidizing agent diamide, the disulfide-reducing agent dithiothreitol, hyper- and hypo-osmotic shock, amino acid starvation, nitrogen source depletion, and progression into stationary phase. A large set of genes (approximately 900) showed a similar drastic response to almost all of these environmental changes. Additional features of the genomic responses were specialized for specific conditions. Promoter analysis and subsequent characterization of the responses of mutant strains implicated the transcription factors Yap1p, as well as Msn2p and Msn4p, in mediating specific features of the transcriptional response, while the identification of novel sequence elements provided clues to novel regulators. Physiological themes in the genomic responses to specific environmental stresses provided insights into the effects of those stresses on the cell. Study is described in more detail in Gasch AP et al.(2000) Mol Biol Cell 11:4241-57 Keywords: other
Project description:Cell growth rate is regulated in response to resource availability including the abundance, and molecular form, of essential nutrients. In the model eukaryotic cell, Saccharomyces cerevisiae (budding yeast), the molecular form of environmental nitrogen impacts both cell growth rate and mRNA expression. Disentangling causal relationships between nitrogen availability, cell growth rate and differential gene expression poses a considerable challenge. Using experimental control of cell growth rate using chemostats, we studied the effect of variation in environmental nitrogen on differential gene expression. We find that the primary determinant of nitrogen-regulated gene expression is nitrogen abundance whereas variation in nitrogen source affects the expression of only a small number of transcripts with highly specialized functions. To study the dynamics of nitrogen-responsive gene expression we perturbed steady-state nitrogen-limited chemostat cultures by addition of either proline or glutamine. Addition of either proline or glutamine to cells growing in nitrogen-limited chemostats results in repression of the nitrogen catabolite repression (NCR) regulon consistent with nitrogen abundance, and not nitrogen source, being the primary determinant of nitrogen-regulated gene expression. We find that a transition from nitrogen-limited to nitrogen-replete conditions is accompanied by rapid induction of transcripts required for protein translation. We identified a reciprocal relationship between specific regulons required for protein translation (RP and RiBi) and the NCR regulon. Using mathematical modeling we find evidence that cells adopt a metabolically inefficient growth mode during this transition. By means of high resolution time series analysis we find evidence that rapid, and potentially accelerated, mRNA degradation plays an important role in remodeling gene expression programs in response to change in environmental nitrogen. We propose that the evolutionarily conserved TORC1 signaling pathway orchestrates the balance between protein translation and assimilation of nitrogen sources at the transcriptional level to optimize rates of cell proliferation. A total of of 102 samples were analyzed in different nitrogen-limited conditions using chemostats in both steady-state and dynamic conditions. A common reference obtained from an ammonium-limited chemostat growing at a dilution rate of 0.12/hr was used for all two color hybridization experiments.
Project description:Purpose: We explore gene expression changes when Neurospora crassa wild type responds to different carbon sources in Vogel's medium. Method: We obtained mRNA samples of Neurospora crassa WT in Vogel's minimal medium (VMM) with different carbon source and used RNA-seq technique to measure the trancriptome changes. Results: We identified many genes of transcription factors and enzymes that were up regulated or down regulated in response to the different carbon stimulation. Conclusion: Our data represents a systematic transcriptome profiling of filamentous fungi on different carbon source and identify COL-26 as a critical regulator in degradation of starch components. Overall design: mRNA profiles of cultures of WT after 4 hours of exposure to media of interest were generated by deep sequencing, in triplicate, using Illumina HiSeq2000. Soluble carbon sources are 2 mM and insoluble carbon source are 2% (w/v).
Project description:In Trypanosoma brucei, most mitochondrial mRNAs undergo U-insertion/deletion editing, and 3′ adenylation and uridylation. The internal sequence changes and terminal extensions are coordinated: Pre-editing addition of the short (A) tail protects the edited transcript against 3′-5′ degradation, while post-editing A/U-tailing renders mRNA competent for ribosome recruitment. Participation of a poly(A) binding protein (PABP) in coupling of editing and 3′ modification processes has been inferred, but its identity and mechanism of action remained elusive. We report identification of KPAF4, a pentatricopeptide repeat-containing PABP which sequesters the A-tail and impedes exonucleolytic degradation. Conversely, KPAF4 inhibits uridylation of A-tailed transcripts and, therefore, premature A/U-tailing of partially-edited mRNAs. This quality check point prevents translation of incompletely edited mRNAs. Our findings also implicate the RNA editing substrate binding complex (RESC) in mediating the interaction between the 5′-end bound pyrophosphohydrolase MERS1 and 3′-end associated KPAF4 to enable mRNA circularization. This event is critical for transcript stability during the editing process.
Project description:Mycobacterium smegmatis is a soil bacterium exposed to continuously environmental changes of nitrogen availability. As member of Actinobacteria, it possesses an ubiquitin-like post-translational modification pathway called pupylation. The pathway starts when a small protein called Pup is attached to a lysine of a specific cellular target. Then the Pup-modified protein is degraded by the proteasome, as it was shown that Pup acts as degradation signal. Recent studies showed the role of pupylation in Mycobacterium smegmatis for survival under nitrogen starvation by supplying recycled amino acids. The present study is an investigation of the influence of Mycobacterium smegmatis Pup protein on the whole proteome in absence of nitrogen sources. Therefore a pup deletion mutant was generated. Applying stable isotope dimethyl labelling, low impact of the pupylation on proteome was revealed immediately after exposure to growth medium lacking nitrogen. In contrast, post 24 h of nitrogen starvation, Msm pup deletion strain showed several proteins with significant changes in abundance. Noteworthy, key proteins involved in nitrogen assimilation were significantly affected in Msm Δpup. Furthermore, we label-free quantified pupylated proteins of nitrogen starved Msm for a more extensive understanding of pupylation role in surviving and overcoming the lack of nitrogen.
Project description:Multiple sclerosis (MS) is a complex disease influenced by genetic and environmental contributing factors. Endocrine disrupting compounds (EDCs) such as bisphenol A (BPA) affect gene expression and hormone-regulated systems throughout the body. We investigated the effects of BPA on Theiler’s-virus induced demyelination (TVID), a mouse model of MS. Perinatal BPA exposure, combined with viral infection, resulted in a decreased level of viral antibodies, accelerated the onset of TVID symptoms, increased inflammation in both the spinal cord and digestive tract, and amplified immune-related gene expression changes induced by viral infection. These results demonstrate the effect of BPA on the trajectory of TVID, and illustrate how multiple factors collectively influence autoimmune disease. For microarray: 7 samples
Project description:The study on the effect of nitrogen replenishment on the gene expression showed that 390 genes were significantly upregulated (with a logFC ranging from 4.5 to 0.8), among which those related to RNA binding, ribosome binding, structural constituent of ribosome, transferase activity and transcription. 833 genes were significantly down-regulated (with a logFC ranging from -0.8 to -5.2) including those related to stress response, amino-acid catabolism process, protein catabolic process (ubiquitin-dependent and vacuolar protein degradation). But the most interesting and unexpected changes were those related to the down-regulation of the glycolytic transcripts. In fact, we observed a rapid down-regulation of many glycolytic mRNA (25/34) 30 min after nitrogen repletion. These data were surprising as expression of the glycolytic genes are generally considered as stable and sometimes they are even used as housekeeping control genes. In addition, the decoupling of the glycolytic gene expression from the expression of RP genes, ribosomal genes or those related to protein synthesis is unique to the present dataset as in most other data expression of these groups are tightly correlated when nutrients are available. This prompts us to examine in some details the factors which led to the control of the glycolytic mRNA instability in conditions of nitrogen replenishment. Overall design: Analysis used RNA collected at 30 min of the beginning of the alcoholic fermentation of S288C with NH4 addition and S288C with H2O addition (control). Experiments made in duplicates - dye-swap design