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Gene expression profiling of human DLD-1 cells after block of CRM1 receptor with Leptomycin B inhibitor.

ABSTRACT: We used RNA-seq approach to define a contribution of mammalian export receptor CRM1 (Chromosomal Maintenance 1 or Exportin 1) into Tpr-dependent export of RNA molecules from the nucleus to the cytoplasm. Basket nucleoporin Tpr was AID-tagged and depleted through Auxin Induced Degradation system. Loss of Tpr led to rapid changes in gene expression profile, and gene expression changes were compared to GANP, NXF1, RanGAP1 (GSE132363) and CRM1 loss and inhibition, respectively. Altogether, we found a minimal subset of RNAs that are controlled both by Tpr nucleoporin and CRM1 receptor.

ORGANISM(S): Homo sapiens

PROVIDER: GSE148560 | GEO | 2020/06/15

REPOSITORIES: GEO

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Gene expression profiling of human DLD-1 cells after instant depletion of basket nucleoporins

Project description:We used RNA-seq approach to distinguish roles of basket nucleoporins Nup153, Nup50, and Tpr in transcription and nucleocytoplasmic trafficking. AID-mediated depletion of BSK-NUPs caused rapid and drastic changes in transcriptomic profiles within two hours of auxin addition. Changes upon Tpr nucleoporin loss were distinct from Nup153 and Nup50, but similar to changes upon loss of export factor NXF1 and GANP subunit of the TREX-2 mRNA export complex. Altogether, we demonstrated a unique role of Tpr nucleoporin in regulating genes expression through mRNA export.

2020-06-04 | GSE132363 | GEO

Nascent RNA-seq of human AID-NeonGreen-Tpr tagged DLD-1 cells after loss of Tpr nucleoporin

Project description:We used SLAM-seq approach to define a contribution of RNA synthesis into RNA abundance changes after Tpr loss. Basket nucleoporin Tpr was AID-tagged and depleted through Auxin Induced Degradation system. Loss of Tpr led to rapid changes in rates of RNA synthesis. The majority of transcripts were downregulated. Analysis of RNA-seq and SLAM-seq indicated that the changes in RNA abundance of most of Tpr-dependent up- and downregulated genes resulted from increased or decreased rates of RNA synthesis, respectively.

2020-06-15 | GSE149890 | GEO

Exportin Crm1 is repurposed as a docking protein to generate microtubule organizing centers at the nuclear pore

Project description:Non-centrosomal microtubule organizing centers (MTOCs) are important for microtubule organization in many cell types. In fission yeast Schizosaccharomyces pombe, the protein Mto1, together with partner protein Mto2 (Mto1/2 complex), recruits the g-tubulin complex to multiple non-centrosomal MTOCs, including the nuclear envelope (NE). Here, we develop a comparative-interactome mass spectrometry approach to determine how Mto1 localizes to the NE. Surprisingly, we find that Mto1, a constitutively cytoplasmic protein, docks at nuclear pore complexes (NPCs), via interaction with exportin Crm1 and cytoplasmic FG-nucleoporin Nup146. Although Mto1 is not a nuclear export cargo, it binds Crm1 via a nuclear export signal-like sequence, and docking requires both Ran in the GTP-bound state and Nup146 FG repeats. In addition to determining the mechanism of MTOC formation at the NE, our results reveal a novel role for Crm1 and the nuclear export machinery in the stable docking of a cytoplasmic protein complex at NPCs.

2018-05-22 | PXD008334 | Pride

Genome-wide mapping of Nup98HoxA9- and Crm1-binding sites in ES cell lines.

Project description:We used ChIP-seq to examine the genome-wide binding of Nup98-HoxA9 and Crm1 in ES cells. Our analysis revealed that Nup98-HoxA9 is highly and preferentially targeted to specific chromatin sites, particularly near Hox cluster regions where the export factor Crm1 is originally prebound. chromatin immunoprecipitation

2016-01-04 | E-GEOD-68783 | biostudies-arrayexpress

A heterochromatin-specific RNA export pathway facilitates piRNA production

Project description:PIWI-interacting RNAs (piRNAs) guide transposon silencing in animals. The 22-30nt piRNAs are processed in the cytoplasm from long non-coding RNAs. How piRNA precursors, which often lack RNA processing hallmarks of export-competent transcripts, achieve nuclear export is unknown. Here, we uncover the RNA export pathway specific for piRNA precursors in the Drosophila germline. This pathway requires Nxf3-Nxt1, a variant of the hetero-dimeric mRNA export receptor Nxf1-Nxt1. Nxf3 interacts with UAP56, a nuclear RNA helicase essential for mRNA export, and CG13741/Bootlegger, which recruits Nxf3-Nxt1 and UAP56 to heterochromatic piRNA source loci. Upon RNA cargo binding, Nxf3 achieves nuclear export via the exportin Crm1, and accumulates together with Bootlegger in peri-nuclear nuage, suggesting that after export, Nxf3-Bootlegger delivers precursor transcripts to the piRNA processing sites. Our findings indicate that the piRNA pathway bypasses nuclear RNA surveillance systems to achieve export of heterochromatic, unprocessed transcripts to the cytoplasm, a strategy also exploited by retroviruses.

2019-03-28 | GSE126578 | GEO

Genome-wide mapping of Nup98HoxA9- and Crm1-binding sites in ES cell lines.

2016-01-04 | GSE68783 | GEO

CRM1 Exportome and nucleocytoplasmic partition

Project description:CRM1 mediates one of the major nuclear export pathways with the broadest range of cargoes. So far, more than 100 structurally and functionally diverse CRM1 cargoes have been described. We employed affinity purification mass spectrometry for in depth characterization of CRM1 "exportome" in three model systems and we identified surprisingly large numbers, namely >700 export substrates from the yeast S. cerevisiae, ≈ 1000 from Xenopus oocytes and >1050 from human cells.I. Futher, we quantified the partitioning of ≈9600 proteoforms between nucleus and cytoplasm of Xenopus oocytes.

2016-01-04 | PXD002899 | Pride

CRM1 inhibitor anti-tumor activity is enhanced with salicylates by S-phase arrest and impaired DNA-damage repair

Project description:Chromosome region maintenance protein1 (CRM1) mediates protein export from the nucleus and is a new target for anti-cancer therapeutics. Broader application of KPT-330 (Selinexor), a CRM1 inhibitor approved for myeloma, has been limited by toxicity. We found that combining choline salicylate (CS) with low-doses of KPT-330 (K+CS) had potent synergistic activity across blood and solid organ cancers ex-vivo and in-vivo. Mechanistically, K+CS enhances CRM1 degradation, induces potent inhibition of CRM1 mediated nuclear export, and arrests cells in S-phase with simultaneous reduction of Rad51 causing impaired DNA damage repair. K+CS represents a potential new class of therapy for multiple cancer types.

2020-10-28 | GSE160186 | GEO

TPR Knockdown Frac-Seq

Project description:How splicing regulates the nuclear export of mRNAs has been a source of much debate. While splicing has been shown to enhance nuclear export, it has remained unclear whether mRNAs generated from intronless genes use specific machinery to promote their export. Here we investigate the role of the major nuclear pore basket protein, TPR, in regulating mRNA and lncRNA nuclear export. We provide evidence that TPR is required for the nuclear export of mRNAs and lncRNAs that are generated from intronless or intron-poor genes. In contrast, TPR is not required for the nuclear retention of mRNAs that have retained introns, or unused 5’ splice site motifs. In summary, our study provides one of the first examples of a factor that is specifically required for the nuclear export of intronless mRNAs.

2020-10-02 | GSE135537 | GEO

A heterochromatin specific RNA export pathway facilitates piRNA production

Project description:PIWI-interacting RNAs (piRNAs), a class of small RNAs that guide transposon silencing in animals, are processed in the cytoplasm from RNA Polymerase II transcripts. How piRNA precursors, which often lack RNA maturation signatures and thereby violate quality control checkpoints, achieve nuclear export is unknown. Here, we uncover a germline-specific RNA export pathway in Drosophila, that escorts piRNA precursors from their heterochromatic origins to nuage, the cytoplasmic piRNA processing centres. This pathway connects canonical nuclear export factors—the RNA helicase UAP56, the NXF cofactor Nxt1/p15, and the exportin Crm1/Xpo1—with Nxf3, a variant of the mRNA exporter Nxf1/Tap. Nxf3 recruitment to nascent piRNA precursors occurs via the heterochromatin protein 1-variant Rhino and CG13741/Bootlegger, a new piRNA pathway factor, thereby making piRNA precursor export independent of RNA processing events. Thus, similar to retroviral hijacking of cellular export factors, piRNA precursor export evolved to bend canonical gene expression rules through bypassing nuclear RNA surveillance mechanisms.

2019-03-29 | PXD012717 | Pride

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