Project description:Transcription is a major obstacle for replication fork progression and a cause of genome instability. Yra1 is an essential nuclear factor of the evolutionarily conserved family of hnRNP-like export factors that when overexpressed impairs mRNA export and cell growth. Through this ChIP-chip analysis it is shown that Yra1 binds to active chromatin and is enriched at telomeres when it is overexpressed, in agreement with a possible role of this mRNP factor in the maintenance of telomere integrity. Our data indicate that YRA1 overexpression correlates with replication impairment as inferred by the increase of Rrm3, a helicase involved in the replication fork progression, at transcribed genes and telomeres. ChIP-chip studies were perfomed with antibodies against HA-tagged Yra1 protein in wild-type cells and cells overexpressing YRA1 of the yeast S. Cerevisiae, as well as Flag-tagged Rrm3 protein in both wild-type and cells overexpressing YRA1.
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:Transcription is a major contributor to genome instability.A main cause of transcription-associated instability relies on the capacity of transcription to stall replication. Such genome instability is increased in RNAPII mutants. ChIP-chips performed in asynchronous cultures showed an increase of the Rrm3 binding signal all over the genome in rpb1-1 compared to wild-type. ChIP-chip studies were perfomed with antibody against Flag-tagged Rrm3 protein in both wild-type and rpb1-1 cells.
Project description:Transcription is a major obstacle for replication fork progression and a cause of genome instability. Such instability increases in mutants with a suboptimal assembly of the nascent messenger ribonucleo-protein particle (mRNP), as THO/TREX and the NPC-associated THSC/TREX-2 complex. Here we show that yeast sac3M-bM-^HM-^F and thp1M-bM-^HM-^F cells accumulate genome-wide replication obstacles as determined by the distribution of the Rrm3 helicase. Such obstacles preferentially occur at long and highly expressed genes, to which Sac3 and its interacting partner Thp1 are preferentially bound in wild-type cells. ChIP-chip studies were perfomed with antibodies against Flag-tagged Thp1 and Sac3 proteins in wild-type cells of the yeast S. Cerevisiae, as well as Flag-tagged Rrm3 protein in sac3M-bM-^HM-^F and thp1M-bM-^HM-^F cells that were compared with Rrm3 in wild-type cells from Santos-Pereira et al., 2013 (accession number GSE50185).