Translation profiling of Schizosaccharomyces pombe size separated polysome fractions provides data on average numbers of associated ribosomes for most transcripts
ABSTRACT: To obtain translational profiles for all mRNAs, polysome preparations are separated according to their size using a sucrose gradient and the mRNAs in each fraction are identified and quantified with DNA microarrays. Starting with exponentially growing cells, we analyzed 12 polysome fractions using DNA microarrays containing elements for all known and predicted genes of fission yeast. This approach provided data on average numbers of associated ribosomes for most transcripts.
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:To obtain global data on polyadenylation of mRNAs, we fractionated the mRNAs according to their poly(A) tail length using a poly-U sepharose column followed by differential elution at five temperatures. Five mRNA fractions with distinct ranges of poly(A) tail length were then hybridized to microarrays using total eluate as a reference.
Project description:The assembly of nucleosomes by histone chaperones is an important component of transcriptional regulation. Here we have assessed the global roles of the S. pombe HIRA histone chaperone complex. Microarray analysis indicates that inactivation of the HIRA complex results in increased expression of at least 4% of fission yeast genes. HIRA-regulated genes overlap with those which are normally repressed in vegetatively growing cells, such as targets of the Clr6 histone deacetylase and silenced genes located in subtelomeric regions. HIRA is also required for silencing of all 13 intact copies of the Tf2 long terminal repeat (LTR) retrotransposon. However, the role of HIRA is not restricted to bona fide promoters, because it also suppresses non-coding transcripts from solo LTR elements and spurious antisense transcripts from cryptic promoters associated with transcribed regions. Furthermore, the HIRA complex is essential in the absence of the quality control provided by nuclear exosome-mediated degradation of illegitimate transcripts. This suggests that HIRA restricts genomic accessibility, and, consistent with this, the chromosomes of cells lacking HIRA are more susceptible to genotoxic agents that cause double strand breaks. Thus the HIRA histone chaperone is required to maintain the protective functions of chromatin.
Project description:SWI/SNF chromatin remodeling complexes play critical roles in transcription and other chromatin-related processes. The analysis of the two members of this class in Saccharomyces cerevisiae, SWI/SNF and RSC, has heavily contributed to our understanding of these complexes. To understand the in vivo functions of SWI/SNF and RSC in an evolutionarily distant organism, we have characterized these complexes in Schizosaccharomyces pombe. While core components are conserved between the two yeasts, the compositions of S. pombe SWI/SNF and RSC differ from their S. cerevisiae counterparts and in some ways are more similar to metazoan complexes. Furthermore, several of the conserved proteins, including actin-like proteins, are strikingly different between the two yeasts with respect to their requirement for viability. Finally, phenotypic and microarray analyses identified widespread requirements for SWI/SNF and RSC on transcription including strong evidence that SWI/SNF directly represses iron transport genes.
Project description:The SAGA complex is a conserved, multifunctional coactivator that plays broad roles in eukaryotic transcription. Previous studies suggested that Tra1, the largest SAGA component, is required either for SAGA assembly or for recruitment by DNA-bound transcriptional activators. In contrast to S. cerevisiae and mouse, a tra1? mutant is viable in S. pombe, allowing us to test these issues in vivo. We find that, in a tra1? mutant, SAGA assembles and is recruited to some, but not all promoters. Consistent with these findings, Tra1 regulates the expression of only a subset of SAGA-dependent genes. We previously reported that the SAGA subunits Gcn5 and Spt8 have opposing regulatory roles during S. pombe sexual differentiation. We show here that, like Gcn5, Tra1 represses this pathway, although by a distinct mechanism. Thus, our study reveals that Tra1 has specific regulatory roles, rather than global functions, within SAGA.
Project description:Spt6 is a conserved factor, critically required for several transcription and chromatin related processes. We now show that Spt6 and its binding partner, Iws1, are required for heterochromatic silencing in Schizosaccharomyces pombe. Our studies demonstrate that Spt6 is required for silencing of all heterochromatic loci and that an spt6 mutant has an unusual combination of heterochromatic phenotypes compared to previously studied silencing mutants. Unexpectedly, we find normal nucleosome positioning over heterochromatin and normal levels of histone H3K9 dimethylation. However, we also find greatly reduced levels of H3K9 trimethylation, elevated levels of H3K14 acetylation, and reduced recruitment of several silencing factors. Our evidence suggests that Spt6 plays a role at both the transcriptional and post-transcriptional levels; in an spt6 mutant, RNA polymerase II (RNAPII) occupancy at the pericentric regions is only modestly increased, while production of small interfering RNAs (siRNAs) is lost. Taken together, our results suggest that Spt6 is required for multiple steps in heterochromatic silencing by controlling chromatin, transcriptional, and post-transcriptional processes.
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.