Transcription profiling of logarithmically growing fission yeast
ABSTRACT: To estimate mRNA steady-state levels we used RNA extracted from logarithmically growing fisson yeast cells on Affymetrix Yeast 2.0 Genechip arrays. The signal intensities from two independent biological repeats were averaged, resulting in measurements for 4818 out of 4962 nuclear protein-coding genes.
Gene expression is controlled at multiple layers, and cells may integrate different regulatory steps for coherent production of proper protein levels. We applied various microarray-based approaches to determine key gene-expression intermediates in exponentially growing fission yeast, providing genome-wide data for translational profiles, mRNA steady-state levels, polyadenylation profiles, start-codon sequence context, mRNA half-lives, and RNA polymerase II occupancy. We uncovered widespread and ...[more]
Project description:General discard pathways eliminate unprocessed and irregular pre-mRNAs to control the quality of gene expression. In contrast to such general pre-mRNA decay, we describe here a nuclear pre-mRNA degradation pathway that controls the expression of select intron-containing genes. We show that the fission yeast nuclear poly(A)-binding protein, Pab2, and the nuclear exosome subunit, Rrp6, are the main factors involved in this polyadenylation-dependent pre-mRNA degradation pathway. Transcriptome analysis and intron swapping experiments revealed that inefficient splicing is important to dictate susceptibility to Pab2-dependent pre-mRNA decay. We also show that negative splicing regulation can promote the poor splicing efficiency required for this pre-mRNA decay pathway, and in doing so identify a mechanism of cross-regulation between paralogous ribosomal proteins through nuclear pre-mRNA decay. Our findings unveil a layer of regulation in the nucleus in which the turnover of specific pre-mRNAs, besides the turnover of mature mRNAs, is used to control gene expression.
Project description:Data on absolute molecule numbers are rare but will empower the modeling, understanding and comparison of cellular functions and biological systems. We quantified transcriptomes and proteomes in fission yeast during both proliferation and quiescence. This rich resource provides the first comprehensive reference for all RNA and most protein concentrations in a eukaryote under key physiological conditions. This integrated dataset will support quantitative biology and afford unique biological insights into cell regulation. While mRNAs are typically expressed in a narrow range above 1 copy/cell, most long non-coding RNAs, except for a specific subset, are strongly repressed below 1 copy/cell. Cell cycle-regulated transcription tunes mRNA numbers to phase-specific requirements but can also lead to switch-like expression. Proteins greatly exceed mRNAs in abundance and dynamic range, and their numbers scales with functional demands. Upon transition to quiescence, the proteome composition changes substantially but, in stark contrast to mRNAs, proteins do not uniformly decrease but scale with cell volume.
Project description:To examine whether naturally occurring duplications are altering gene expression we chose eight pairs of closely related strains (less than 150 SNPS between each pair) that contained at least one unshared duplication and performed pair-wise two-colour microarray analysis.
Project description:Gene expression is controlled globally and at multiple levels in response to environmental stress, but the relationships among these dynamic regulatory changes are not clear. Here we analyzed genome regulation in response to stress in the fission yeast, Schizosaccharomyces pombe, combining dynamic genome-wide data on mRNA levels and translational profiles with data on the levels of 2148 proteins (43% of proteome).
Project description:The SAGA complex is a conserved multifunctional coactivator known to play broad roles in eukaryotic transcription. To gain new insights into its functions, we have performed biochemical and genetic analyses of SAGA in the fission yeast, Schizosaccharomyces pombe. Purification of the S. pombe SAGA complex showed that its subunit composition is identical to that of Saccharomyces cerevisiae. Analysis of S. pombe SAGA mutants revealed that SAGA has two opposing roles regulating sexual differentiation. First, in nutrient rich conditions, the SAGA histone acetyltransferase, Gcn5, represses ste11+, which encodes the master regulator of the mating pathway. In contrast, the SAGA subunit Spt8 is required for the induction of ste11+ upon nutrient starvation. Chromatin immunoprecipitation experiments suggest that these regulatory effects are direct, as SAGA is physically associated with the ste11+ promoter independent of nutrient levels. Genetic tests suggest that nutrient levels do cause a switch in SAGA function, as spt8? suppresses gcn5? with respect to ste11+ derepression in rich medium, whereas the opposite relationship, gcn5? suppression of spt8?, occurs during starvation. Thus, SAGA plays distinct roles in the control of the switch from proliferation to differentiation in S. pombe through the dynamic and opposing activities of Gcn5 and Spt8.