Project description:Crosslinked DNA from wild type cells or from mutant of the THO complex was analyzed by tiling arrays to precisely detect DNA targets of THO in yeast.
Project description:Crosslinked DNA from wild type cells or from mutant of the THO complex was analyzed by tiling arrays to precisely detect DNA targets of THO in yeast. DNA coming from SDS-washed DCF pellet and the supernatant SN2k of 2 strains were compared in the study, WT (W303) and mft1 delta (DLY224). The DNA of each condition (Pellet or Supernatant) was labeled either with Cy3 or Cy5 fluorescent dyes and competitively hybridized on 244k agilent arrays. For each strain, the experiment was performed on 2 biological replicates, with dye swap.
Project description:THO/TREX is a conserved nuclear complex that functions in mRNP biogenesis and prevents transcription-associated recombination. Whether or not it has a ubiquitous role in the genome is an open question. ChIP-chip studies reveal that the Hpr1 component of THO and the Sub2 RNA-dependent ATPase have genome wide-distributions at active ORFs in yeast. In contrast to RNAPII, evenly distributed from promoter to termination regions, THO and Sub2 are absent at promoters and distributed in a sharp 5’→3’ gradient. Importantly, ChIP-chips reveal an over-recruitment of Rrm3 in active genes in THO mutants that is reduced by overexpression of RNase H1. Our work establishes a genome-wide function for THO-Sub2 in transcription elongation and mRNP biogenesis that function to prevent the accumulation of transcription-mediated replication obstacles, including R-loops.
Project description:THO/TREX is a conserved nuclear complex that functions in mRNP biogenesis and prevents transcription-associated recombination. Whether or not it has a ubiquitous role in the genome is an open question. ChIP-chip studies reveal that the Hpr1 component of THO and the Sub2 RNA-dependent ATPase have genome wide-distributions at active ORFs in yeast. In contrast to RNAPII, evenly distributed from promoter to termination regions, THO and Sub2 are absent at promoters and distributed in a sharp 5M-bM-^@M-^YM-bM-^FM-^R3M-bM-^@M-^Y gradient. Importantly, ChIP-chips reveal an over-recruitment of Rrm3 in active genes in THO mutants that is reduced by overexpression of RNase H1. Our work establishes a genome-wide function for THO-Sub2 in transcription elongation and mRNP biogenesis that function to prevent the accumulation of transcription-mediated replication obstacles, including R-loops. ChIP-chip studies were perfomed with tagged forms of the Hpr1 component of THO (Hpr1-FLAG), the Sub2 RNA-dependent ATPase of TREX (Sub2-FLAG), the Rpb3 subunit of RNA polymerase II (Rpb3-PK) and the Rrm3 protein (Rrm3-FLAG) in the yeast S. cerevisiae.
Project description:Changes in RNA levels during osmotic stress were investigated. Total RNA was extracted from a wild-type yeast strain before and after treatment with 0.4 M NaCl and the corresponding cDNAs were hybridazed on Tiling arrays. In particular, for all the intron-containing genes, the changes in the levels of intron signal in stressed cells related to the intron signal in the non-stressed cells, and the changes in the levels of exon signal in stresses cells related to the exon signal in non-stressed cells were investigated. The supplementary bar file contains the ratios between stress signals respect to non-stress signals, using the average of the 3 biological replicas.
Project description:Changes in RNA levels during osmotic stress were investigated. Total RNA was extracted from a wild-type yeast strain before and after treatment with 0.4 M NaCl and the corresponding cDNAs were hybridazed on Tiling arrays. In particular, for all the intron-containing genes, the changes in the levels of intron signal in stressed cells related to the intron signal in the non-stressed cells, and the changes in the levels of exon signal in stresses cells related to the exon signal in non-stressed cells were investigated. The supplementary bar file contains the ratios between stress signals respect to non-stress signals, using the average of the 3 biological replicas. For the tiling array experiments, total RNA was extracted from exponentially YPD growing W303-1A cells before and after treatment for 15 min with 0.4M NaCl. The hybridization of tiling array Affymetrix (GeneChipM-BM-. S. cerevisiae Tiling 1.0R Array) with cDNA produced from total RNA, was carried out according to the Affymetrix protocol and using GeneChipM-BM-. Hybridization, Wash and Stain Kit (P/N 900720). The final data were obtained from the results of three independent biological experiments. High-density tiling arrays images were normalised and analysed using the Affymetrix Tiling Analysis Software; and were visualized by the Integrated Genome Browser (Affymetrix).
Project description:THO/TREX is a conserved nuclear complex that functions in mRNP biogenesis at the interface of transcription-RNA export with a key role in preventing transcription-associated genome instability. We used microarrays to analyze the impact of different THO/TREX mutations on gene expression and found that THO-Sub2 deletions have a high functional impact on highly expressed, long and G+C-rich genes regardless of gene function.