Cancer-associated SF3B1 Mutations Inhibit mRNA Nuclear Export by Disrupting SF3B1–THOC5 Interactions
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PROVIDER: PRJNA1116549 | ENA |
REPOSITORIES: ENA
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Project description:Pre-mRNA splicing and nuclear export are coordinated processes that ensure efficient and accurate gene expression. In recent years, disruptions in this process have been found to result in substantial gene expression aberrations, with potential contributions from SF3B1 mutations, whose mechanism remains elusive. The present study reveals that the K700E mutation in SF3B1 attenuates its interaction with THOC5, resulting in reduced mRNA binding by THOC5, and ultimately, the inhibition of mRNA nuclear export. Interestingly, overexpressing THOC5 can restore the attenuated interaction, facilitating mRNA nuclear export. Importantly, other cancer-associated SF3B1 mutations may exert a similar impact. Our research highlights the critical role of the THOC5–SF3B1 interaction in regulating mRNA nuclear export and provides valuable insights into the impact of SF3B1 mutations on this process.
2024-09-12 | GSE268355 | GEO
Project description:SF3B1 mutations are the most frequent spliceosomal alterations across cancers, yet no successful therapy exists to target this pathway. Previous findings from a phase 2 clinical trial of the XPO1 inhibitor selinexor in patients with high-risk myelodysplastic neoplasms (MDS) relapsed or refractory to hypomethylating agents (HMA) revealed increased activity in patients with SF3B1 mutations. XPO1 (Exportin-1) is responsible for the export of over 200 proteins, but also plays a role in the transport of multiple RNA species, including small nuclear RNAs (snRNAs), ribosomal RNAs (rRNAs), and select messenger RNAs (mRNAs) out of the nucleus. We therefore hypothesized that XPO1 inhibition perturbs RNA export and may preferentially affect SF3B1 mutants via altered splicing, given the role of XPO1 in exporting snRNAs, which form the catalytic portion of the spliceosome. To evaluate the mechanism underlying preferentially sensitivity of SF3B1-mutants to XPO1 inhibition, we performed nuclear and cytoplasmic fraction followed by RNA sequencing before and after XPO1 inhibition in SF3B1 wildtype and SF3B1 K666N cells (subcellular RNA-seq). Whole transcriptomic analysis of subcellular RNA-seq data revealed more nuclear retention of global RNA transcripts after XPO1 inhibition in the SF3B1 mutant cell line. Similarly, we performed subcellular RNA-seq for small RNAs and found snRNAs to be increased in the nucleus after XPO1 inhibition in the SF3B1 mutant cells. We then performed total cellular RNA sequencing to understand the effect of XPO1 inhibition on global RNA expression and RNA splicing. Differential gene expression analysis identified that XPO1 inhibition had the greatest effect on cell cycle in SF3B1 wildtype cells but in SF3B1-mutant cells differentiation pathways were more significantly affected. Alternative splicing analysis showed increased 3’ alternative splicing events in SF3B1 mutant after XPO1 inhibition. These results signify the mechanistic basis for preferential sensitivity of SF3B1 mutant cells to nuclear export inhibition arises through nuclear retention of spliceosomal snRNAs and select mRNAs that result in perturbation of differentiation pathways.
2024-08-05 | GSE255179 | GEO
Project description:Altered RNA export by SF3B1 mutants confers sensitivity to nuclear export inhibition
| PRJNA1073893 | ENA
Project description:SF3B1, which encodes an essential spliceosomal protein, is frequently mutated in myelodysplastic syndromes (MDS) and many cancers. However, the defect of mutant SF3B1 is unknown. Here, we analyzed RNA-sequencing data from MDS patients and confirmed that SF3B1 mutants use aberrant 3' splice sites. To elucidate the underlying mechanism, we purified complexes containing either wild-type or the hotspot K700E mutant SF3B1, and found that levels of a poorly studied spliceosomal protein, SUGP1, were reduced in mutant spliceosomes. Strikingly, SUGP1 knockdown completely recapitulated the splicing errors, whereas SUGP1 overexpression drove the protein, which our data suggests plays an important role in branchsite recognition, into the mutant spliceosome and partially rescued splicing. Other hotspot SF3B1 mutants showed similar altered splicing and diminished interaction with SUGP1. Our study demonstrates that SUGP1 loss is the sole defect of mutant SF3B1 spliceosomes and, since this defect can be rescued, suggests possibilities for therapeutic intervention.
2019-07-24 | GSE128805 | GEO
Project description:Cancer-Causing Mutations in SF3B1 Alter Splicing by Disrupting Interaction with SUGP1
| PRJNA528926 | ENA
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: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: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