A member of transcription/export complex (TREX), THOC5 controls elongation rate by recruiting CDK12.
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PROVIDER: PRJNA699287 | ENA |
REPOSITORIES: ENA
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Project description:THOC5, a member of the THO complex that is a subcomplex of Transcription/export complex (TREX) 1, is essential for 3´processing of slow kinetics IEGs, and for export of only a subset of genes. Applying nanopore mRNA-sequence technology, we show here that upon depletion of THOC5, 50% of mRNAs showed alteration of 3´end cleavage sites. Moreover, polymerase-II (Pol II)-CHIP-sequencing data reveals that upon depletion of THOC5 Pol II density was increased at gene body and 3´UTR. THOC5 is recruited near to Pol II high density sites except promotor regions, suggesting that THOC5 is involved in transcription elongation. Indeed, 385 THOC5 independent genes in fast Pol II cells became THOC5 dependent in slow Pol II cells. Chromatin associated THOC5 in slow Pol II cells interacts with CDK12, RNA helicases DDX5 and DDX, and THOC6, but not with other members of THO complex. Notably, THOC5 did not form this complex in fast poly II cells. CDK12 was recruited to R-loop (DNA:RNA hybrid) in THOC5/THOC6 dependent manner. Here, THOC6, but not THOC5 directly interacts with R-loop then, recruited DDX5 resolves R-loop and then CDK12 enhances further transcription elongation. These data show for the first time the novel function of THOC5/THOC6 complex during transcription elongation.
2022-12-31 | GSE166115 | GEO
Project description:Hepatocellular carcinoma (HCC) is a frequent cancer with poor prognosis and with limited possibilities for anti-cancer treatment. Evidence has recently accumulated suggesting that multiple signaling pathways are activated in human cancer. It has been recently shown by exosome sequencing of HCC that 161 putative driver genes are associated with 11 recurrently altered pathways, suggesting that we need to inhibit these multiple pathways for HCC therapy. THOC5, a member of transcription/export (TREX) complex, that plays a role in less than 1% of mRNA processing in normal cells such as fibroblasts or macrophages, is not required for maintenance of mature hepatocytes. In this study, we have examined the role of THOC5 in human HCC. Enhanced THOC5 expression was not observed in differentiation grading 1 (G1) human HCC, but 78% of G2 and G3 exhibited increased levels of THOC5. Furthermore, the 50% depletion of THOC5 in hepatocellular carcinoma cell lines, Huh7 and HepG2 causes lipid accumulation and apoptosis. Transcriptome analysis using THOC5 depleted Huh7 and HepG2 cells revealed that 1049 and 837 genes were downregulated upon depletion of THOC5 in Huh7 and HepG2 cells, respectively. Among these genes, 396 were commonly downregulated in both cell lines. Some of these, such as tissue inhibitor of metalloproteinase 3 (TIMP3), tripartite motif containing 24 (TRIM24), ribosomal protein S6 kinase polypeptide 3 (RPS6KA3) and transmembrane emp24-like trafficking protein 10 (TMED10) are known to be fine tuners for several signal transduction pathways. The expression of these proteins was correlated with the THOC5 expression level in HCC. To inhibit multiple signaling pathways we are currently examining the effects of a combination of siRNAs against THOC5 target genes in HCC cell lines. Our data suggest that THOC5, which controls a set of genes that are involved in HCC malignancy can be a potential biomarker for HCC. In addition, THOC5 target gene, TMED10 may also be a novel biomarker of HCC. Furthermore, the suppression of the THOC5 gene per se or multiple THOC5 target genes may represent a novel strategy for cancer therapy. Huh7 and HepG2 cells were infected with lentiviral THOC5 and control shRNAs. Five days after infection, total RNAs were isolated and subjected to microarray analysis.
2015-11-02 | E-GEOD-70178 | biostudies-arrayexpress
Project description:Hepatocellular carcinoma (HCC) is a frequent cancer with poor prognosis and with limited possibilities for anti-cancer treatment. Evidence has recently accumulated suggesting that multiple signaling pathways are activated in human cancer. It has been recently shown by exosome sequencing of HCC that 161 putative driver genes are associated with 11 recurrently altered pathways, suggesting that we need to inhibit these multiple pathways for HCC therapy. THOC5, a member of transcription/export (TREX) complex, that plays a role in less than 1% of mRNA processing in normal cells such as fibroblasts or macrophages, is not required for maintenance of mature hepatocytes. In this study, we have examined the role of THOC5 in human HCC. Enhanced THOC5 expression was not observed in differentiation grading 1 (G1) human HCC, but 78% of G2 and G3 exhibited increased levels of THOC5. Furthermore, the 50% depletion of THOC5 in hepatocellular carcinoma cell lines, Huh7 and HepG2 causes lipid accumulation and apoptosis. Transcriptome analysis using THOC5 depleted Huh7 and HepG2 cells revealed that 1049 and 837 genes were downregulated upon depletion of THOC5 in Huh7 and HepG2 cells, respectively. Among these genes, 396 were commonly downregulated in both cell lines. Some of these, such as tissue inhibitor of metalloproteinase 3 (TIMP3), tripartite motif containing 24 (TRIM24), ribosomal protein S6 kinase polypeptide 3 (RPS6KA3) and transmembrane emp24-like trafficking protein 10 (TMED10) are known to be fine tuners for several signal transduction pathways. The expression of these proteins was correlated with the THOC5 expression level in HCC. To inhibit multiple signaling pathways we are currently examining the effects of a combination of siRNAs against THOC5 target genes in HCC cell lines. Our data suggest that THOC5, which controls a set of genes that are involved in HCC malignancy can be a potential biomarker for HCC. In addition, THOC5 target gene, TMED10 may also be a novel biomarker of HCC. Furthermore, the suppression of the THOC5 gene per se or multiple THOC5 target genes may represent a novel strategy for cancer therapy.
2015-11-02 | GSE70178 | GEO
Project description:N6-methyladenosine (m6A) is the most abundant internal modification of eukaryotic mRNA. This modification has previously been shown to alter the export kinetics for mRNAs though the molecular details surrounding this phenomenon remain poorly understood. Here we show that the m6A complex (WTAP, KIAA1429, METTL3/14) drives recruitment of the TREX mRNA export complex onto m6A modified mRNAs and this process is essential for the efficient export of certain mRNAs. Depletion of the core m6A complex leads to loss of TREX from mRNAs which undergo the m6A modification. We show that TREX stimulates recruitment of the m6A reader protein YTHDC1 to the mRNP and the m6A complex influences the interaction of TREX with YTHDC1. We suggest that m6A acts as a surrogate for other TREX recruitment mechanisms such as splicing and 5’ capping, in long internal and final exons which may otherwise be devoid of this essential complex for mRNA export.
2018-10-03 | GSE111878 | GEO
Project description:THOC5, a member of the THO complex, is essential for the 3´processing of some inducible genes, the export of a subset of genes and stem cell self-renewal. Utilising nanopore mRNA-sequencing we show that when THOC5 is depleted 50-60% of mRNAs undergo altered 3´end cleavage. Further, THOC5 depletion leads to increased RNA Polymerase II (Pol II) presence at the start site, on the gene body and close to the 3’end. Moreover, THOC5 is recruited close to high density Pol II sites suggesting that THOC5 is involved in transcriptional elongation. Indeed, DRB/TTchem-seq that measures elongation rates in vivo revealed an accumulation of released Pol II near to TSS and a decrease of elongation rates in THOC5 depleted cells. Furthermore, THOC5 is more recruited to its target genes in cells expressing slow Pol II than those expressing fast Pol II. In cells expressing slow Pol II chromatin associated THOC5 interacts with CDK12 (a protein that modulates mRNA elongation rates), RNA helicases DDX5, DDX17, and THOC6. Importantly these interactions were not observed in cells expressing fast Pol II. 3’ cleavage of 50% of THOC5 target genes is also regulated by CDK12 and THOC6. The CDK12/THOC5 interaction promotes CDK12 recruitment to R-loops in a THOC6-dependent manner. These data demonstrate that THOC5/THOC6 play a part in transcription elongation. Given the role of THOC5 in primitive cell survival its phosphorylation by agonists, oxidative stress and oncogenes the pathway we identified has relevance in the physiology and pathology of stem cells.
2022-12-16 | GSE173374 | GEO
Project description:THOC5, a member of the THO complex, is essential for the 3´processing of some inducible genes, the export of a subset of genes and stem cell self-renewal. Utilising nanopore mRNA-sequencing we show that when THOC5 is depleted 50% of mRNAs undergo altered 3´end cleavage. Further, THOC5 depletion leads to increased RNA Polymerase II (Pol II) presence on the gene body and close to the 3’end. Moreover, THOC5 is recruited close to high density Pol II sites except those at the promotor regions suggesting that THOC5 is involved in transcriptional elongation. Indeed, THOC5 independent genes in cells expressing fast Pol II became THOC5 dependent in cells expressing slow Pol II. In cells expressing slow Pol II chromatin associated THOC5 interacts with CDK12 (a protein that modulates mRNA elongation rates), RNA helicases DDX5, DDX17, and THOC6. Importantly these interactions were not observed in cells expressing fast Pol II. The CDK12/THOC5 interaction promotes CDK12 recruitment to R-loops in a THOC6-dependent manner. These data demonstrate that THOC5/THOC6 play a part in transcription elongation. Given the role of THOC5 in primitive cell survival its phosphorylation by agonists, oxidative stress and oncogenes the pathway we identified has relevance in the physiology and pathology of stem cells
2022-12-12 | PXD035092 | Pride
Project description:THOC5, a member of the mRNA export complex, is essential for hematopoiesis in vivo and is required for CSF-1 induced macrophage differentiation, thereby playing a key role in the mRNA export of immediate early genes induced by CSF-1 stimulation. Hematopoiesis, growth, differentiation, and commitment to a restricted lineage are guided by a timely expressed set of cytokine receptors and their down-stream transcription factor genes. Transcriptional control mechanisms of gene expression during differentiation were mainly studied by focusing on the cis- and trans-element in promoters, however, the role of mRNA export machinery during differentiation have not been adequately examined. A member of the mRNA export complex, THOC5 which is a substrate for the macrophage-colony stimulating factor (CSF-1) receptor, ATM kinase, or protein kinase C, is an essential element in the maintenance of hematopoiesis in adult mice. Using tamoxifen inducible THOC5 knockout mice, we show here that the depletion of THOC5 impaired myeloid differentiation, but does not influence terminally differentiated organs. Furthermore, an in vitro study showed that the depletion of THOC5 in bone marrow cells results in abnormal CSF-1 induced macrophage differentiation. Transcriptome analysis using cytoplasmic RNA derived from macrophages reveals that only 99 genes were down-regulated 3 days after the depletion of THOC5, however, immediate early genes induced by CSF-1 stimulation, such as Ets family genes, and regulators of myeloid differentiation HoxA1, Id1, Id3 were THOC5 direct target mRNAs, suggesting that THOC5 plays a key role in myeloid differentiation. In each of two biological experiments (called rep1 and rep2 below) six different cytoplasmic RNA samples were generated from bone marrow derived macrophages. These different samples (conditions) correspond to: 1) Cells from ERT2 Cre control mice at day 0, 2) Cells from ERT2 Cre control mice at day 3 of differentiation, 3) Cells from ERT2 Cre control mice at day 3 of differentiation stimulated with tamoxifen, 4) Cells from ERT2 Cre Thoc5 (flox/flox) mice at day 0, 5) Cells from ERT2 Cre Thoc5 (flox/flox) mice at day 3 of differentiation, 6) Cells from ERT2 Cre Thoc5 (flox/flox) mice at day 3 of differentiation stimulated with tamoxifen. Day 0 samples of a given genotype were utilized as common reference in two microarrays and were accordingly co-hydridized against both 3-days differentiated samples (treated or none with tamoxifen) of the same genotype and biological experiment. Thus, four dual-color microarrays from each of the two biological replicate series were performed (giving rise to 8 dual-color microarrays in total). The complete set of microarrays from the second biological experiment (rep2) was performed with inverted labeling directions (dye-swap approach).
2013-12-24 | E-GEOD-41170 | biostudies-arrayexpress
Project description:THOC5, a member of the mRNA export complex, is essential for hematopoiesis in vivo and is required for CSF-1 induced macrophage differentiation, thereby playing a key role in the mRNA export of immediate early genes induced by CSF-1 stimulation. Hematopoiesis, growth, differentiation, and commitment to a restricted lineage are guided by a timely expressed set of cytokine receptors and their down-stream transcription factor genes. Transcriptional control mechanisms of gene expression during differentiation were mainly studied by focusing on the cis- and trans-element in promoters, however, the role of mRNA export machinery during differentiation have not been adequately examined. A member of the mRNA export complex, THOC5 which is a substrate for the macrophage-colony stimulating factor (CSF-1) receptor, ATM kinase, or protein kinase C, is an essential element in the maintenance of hematopoiesis in adult mice. Using tamoxifen inducible THOC5 knockout mice, we show here that the depletion of THOC5 impaired myeloid differentiation, but does not influence terminally differentiated organs. Furthermore, an in vitro study showed that the depletion of THOC5 in bone marrow cells results in abnormal CSF-1 induced macrophage differentiation. Transcriptome analysis using cytoplasmic RNA derived from macrophages reveals that only 99 genes were down-regulated 3 days after the depletion of THOC5, however, immediate early genes induced by CSF-1 stimulation, such as Ets family genes, and regulators of myeloid differentiation HoxA1, Id1, Id3 were THOC5 direct target mRNAs, suggesting that THOC5 plays a key role in myeloid differentiation.
2013-12-24 | GSE41170 | GEO
Project description:The conserved THO/TREX complex is critical for pre-mRNA processing and mRNA nuclear export. In Metazoa, TREX is loaded on nascent RNA transcribed by RNA polymerase II in a splicing-dependent fashion; however, how TREX functions is poorly understood. Here we show that Thoc5 and other TREX components are essential for the biogenesis of piRNA, a distinct class of small non-coding RNAs that control expression of transposable elements (TE) in the Drosophila germline. Mutations in TREX lead to defects in piRNA biogenesis resulting in derepression of multiple TE families, gametogenesis defects, and sterility. TREX components are enriched on piRNA precursors transcribed from dual-strand piRNA clusters and co-localize in distinct nuclear foci that overlap with sites of piRNA transcription. The localization of TREX in nuclear foci and its loading on piRNA precursor transcripts depends on Cutoff, a protein associated with chromatin of piRNA clusters. Finally, we show that TREX is required for accumulation of nascent piRNA precursors. Our study reveals a novel splicing-independent mechanism for TREX loading on nascent RNA and its importance in piRNA biogenesis.
2016-03-18 | GSE79325 | GEO
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.
2020-03-24 | GSE128877 | GEO