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: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: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.
Project description:RNA helicases constitute a large protein family implicated in cellular RNA homeostasis and disease development. Here we show that the RNA helicase Ighmbp2, linked to the neuromuscular disorder SMARD1 (DSMA1) associates with polysomes and impacts on translation of cellular mRNAs containing short, GC-rich and highly structured 5’UTRs. Absence of Ighmbp2 causes ribosome stalling at the start codon of target mRNAs, leading to their reduced translation efficiency. The main mRNA targets of Ighmbp2-mediated regulation encode for components of the THO complex that links mRNA production and nuclear export. Accordingly, failure of Ighmbp2 regulation of the THO complex causes perturbations of the cellular transcriptome and its encoded proteome. Ablation of essential THO complex subunits phenocopies these perturbations. Thus, Ighmbp2 is an upstream regulator of the THO complex that impacts on cellular mRNA homeostasis. Of note, Ighmbp2 dependent regulation of the THO complex is also observed in astrocytes derived from DSMA1 patients, suggesting that de-regulated mRNA metabolism contributes to SMARD1 etiology.
Project description:RNA helicases constitute a large protein family implicated in cellular RNA homeostasis and disease development. Here we show that the RNA helicase Ighmbp2, linked to the neuromuscular disorder SMARD1 (DSMA1) associates with polysomes and impacts on translation of cellular mRNAs containing short, GC-rich and highly structured 5’UTRs. Absence of Ighmbp2 causes ribosome stalling at the start codon of target mRNAs, leading to their reduced translation efficiency. The main mRNA targets of Ighmbp2-mediated regulation encode for components of the THO complex that links mRNA production and nuclear export. Accordingly, failure of Ighmbp2 regulation of the THO complex causes perturbations of the cellular transcriptome and its encoded proteome. Ablation of essential THO complex subunits phenocopies these perturbations. Thus, Ighmbp2 is an upstream regulator of the THO complex that affects cellular mRNA homeostasis. Of note, Ighmbp2 dependent regulation of the THO complex is also observed in astrocytes derived from SMARD1 patients, suggesting that de-regulated mRNA metabolism contributes to SMARD1 etiology and offers new avenues for developing alternative therapeutics to treating SMARD1.
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. S. cerevisiae strains were grown in YPAD liquid culture, total RNA was isolated and hybridized on Affymetrix microarrays.
Project description:RNA helicases constitute a large protein family implicated in cellular RNA homeostasis and disease development. Here we show that the RNA helicase Ighmbp2, linked to the neuromuscular disorder SMARD1 associates with polysomes and impacts on translation of cellular mRNAs containing short, GC-rich and highly structured 5’UTRs. Using UV-crosslinking followed by RNAseq and BS3-crosslinking combined with mass spectrometry we were able to localize Ighmbp2 on ribosomes. Absence of Ighmbp2 causes ribosome stalling at the start codon of target mRNAs, leading to their reduced translation efficiency. The main mRNA targets of Ighmbp2-mediated regulation encode for components of the THO complex that links mRNA production and nuclear export. Accordingly, failure of Ighmbp2 regulation of the THO complex causes perturbations of the cellular transcriptome and its encoded proteome. Ablation of essential THO complex subunits phenocopies these perturbations. Thus, Ighmbp2 is an upstream regulator of the THO complex that affects cellular mRNA homeostasis. Of note, Ighmbp2-dependent regulation of the THO complex is also observed in astrocytes derived from SMARD1 patients, suggesting that de-regulated mRNA metabolism contributes to SMARD1 etiology and offers new avenues for developing alternative therapeutics to treating SMARD1.
Project description:Transcriptional profiling of pear tree comparing a resistant/tolerant cultivar with a susceptible cultivar to the Stemphylium vesicarium fungus Rocha' pear is an economically important portuguese Pyrus communis L. cultivar very susceptible to the Stemphylium vesicarium pathogenic fungus, the brown spot agent, causing huge decrease on fruit quality and yield production. Field control of brown spot disease is based in systemic application of antifungal chemicals with high economic costs and dramatic consequences to public health and environmental pollution. Plant-pathogen interactions involve a series of events encompassing constitutive and induced plant defence responses whose dissection has been a research target for control many crop diseases. The biosynthesis of cell wall polymers and antifungal compounds appear to be an efficient physical and chemical barrier to infection.To understand the molecular responses behind defence mechanisms of resistant/tolerant and susceptible cultivars of Pyrus communis L. to the S. vesicarium fungus, cDNA microarray technology was used to identify the genes differentially expressed along a time course leaf inoculation between 'Rocha' pear cultivar (a high susceptible cultivar) and 'Ercolini' pear cultivar (a resistant/tolerant pear cultivar). This study aims to contribute with information on the molecular mechanisms involved in host-pathogen interactions responsible for pear tree brown spot disease and resistance to Stemphylium vesicarium.
Project description:Transcription termination of mRNAs transcribed from a given locus has a decisive role in regulating the gene function as it determines the coding potential and inclusion of regulatory sequence elements. Failure in appropriate transcription termination leads to read-through transcription, resulting in the synthesis of antisense RNAs which can have profound impact on overall gene expression. However, molecular mechanisms which regulate transcription termination and chimeric RNA formation are poorly understood. We explored the regulatory function of transcription and export complex (THO/TREX) in transcription termination. We show that two members of THO/TREX complex, TREX COMPONENT 1 (TEX1) and HYPER RECOMBINATION1(HPR1) are critical for the correct transcription termination in Arabidopsis. We first demonstrate this by showing defective termination of the bacterial nopaline synthase (NOS) terminator on a transgene in tex1 and hpr1 mutants. Additionally, we show that RNA termination defects in tex1 and hpr1 mutants are widespread at the whole genome levels leading to 3’UTR extensions, truncations and in some cases in the formation of intergenic chimeric transcripts. Chromatin immunoprecipitation coupled with quantitative PCR experiments confirmed the presence of RNA polymerase II beyond the canonical termination sites on genes with defective RNA termination in tex1 and hpr1 mutants. These results demonstrate that THO/TREX complex is a novel regulator of transcription termination in Arabidopsis.