Non-destructive transcriptomics via vesicular export (NTVE)
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ABSTRACT:
PROVIDER: PRJNA1192227 | ENA |
REPOSITORIES: ENA
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Project description:Transcriptomics is a valuable technique for multiplexed monitoring of cellular states. Using an engineered RNA adapter, NTVE exports cellular transcripts from inducible human and murine reporter cell lines with high concordance to conventional lysate-derived RNA-seq. NTVE allows monitoring of transcriptome changes in response to genetic and chemical perturbations (Interferon gamma; ASCL1 transactivation) in the same cells over time using standard sequencing workflows.
2025-09-01 | GSE283136 | GEO
Project description:Palaeoproteomic study of mummified human skin using a non-destructive sampling technique, based on mixed-bed chromatographic media stabilised on ethylene vinyl acetate membranes (“EVA”), which had previously been used exclusively on historical material, was successful in extracting ancient proteins from the surface of Ancient Egyptian mummies. We tested the method on a decontextualised fragment of skin and assessed the endogeneity of its metaproteome by comparison with a procedural blank. Furthermore, we retrieved and authenticated sequences of collagen and keratin from the mummy of a young woman (Supp. 16747 of the Museum of Anthropology and Ethnography of the University of Turin) who lived and died between 2400 and 2200 BC, during the Old Kingdom of Egypt.
2020-06-10 | PXD015572 | Pride
Project description:Vesicular stomatitis virus (VSV): Genome sequencing and assembly
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Project description:Extracellular vesicles (EVs) are lipid bilayered vesicles released into extracellular milieu by most of cells. Currently, various purification methods of EVs have been established but most of studies use ultracentrifugation-based purification of EVs. However, these methods have the problem of co-purification with non-vesicular contaminants including various different types of particles, small apoptotic vesicles, protein aggregates, and other derived from dead cells or abundant extracellular proteins. Therefore, evaluation of non-vesicular contaminants gives valuable information in EV research. Here, we purified EVs with high purity using density gradient ultracentrifugation and then purified EVs were treated by trypsin to discriminate extra-vesicular proteins from intra-vesicular proteins.
2021-08-30 | PXD001355 | Pride
Project description:RNA turnover is critical for regulating gene expression and cellular homeostasis, with nuclear export representing a key step in mRNA fate. During lytic reactivation of Kaposi’s Sarcoma-associated Herpesvirus (KSHV), widespread host mRNA decay is mediated by the viral endonuclease SOX, which depletes cytoplasmic transcripts and induces secondary nuclear RNA processing defects. One such defect includes transcript hyperadenylation, which typically promotes nuclear retention and decay. However, a subset of host mRNAs escapes both SOX-mediated degradation and nuclear retention, raising questions about their export mechanisms and stability. Here, we investigate the impact of KSHV infection on mRNA poly(A) tail length and nuclear export dynamics using high-throughput poly(A)-sequencing of nuclear and cytoplasmic RNA fractions from KSHV-positive cells. Our data confirm a global increase in poly(A) tail length during KSHV lytic infection, consistent with transcript hyperadenylation. Nevertheless, we identified a group of hyperadenylated transcripts that remain cytoplasmically localized, suggesting active evasion of nuclear retention. Notably, we focused on interleukin-6 (IL-6), a known SOX escapee, which retains cytoplasmic expression during lytic infection despite acquiring a longer poly(A) tail. Using G/I tailing and sPAT assays, we confirmed IL-6 hyperadenylation and demonstrate that its export is CRM1-dependent rather than through the canonical NXF1-NXT1 pathway, which is suppressed during host shutoff. Pharmacological inhibition of CRM1 impairs IL-6 nuclear export and reduces steady-state RNA levels, implicating CRM1 export in the stabilization of this transcript. Our findings reveal a previously unrecognized mechanism by which select host mRNAs, including IL-6, bypass KSHV-imposed nuclear export blocks and escape nuclear decay, thereby preserving their function during viral infection. This study highlights viral manipulation of RNA processing and export pathways as a critical determinant of transcript fate and identifies CRM1 as a key mediator of selective host transcript preservation during KSHV lytic reactivation.
2025-05-29 | GSE298531 | GEO
Project description:mRNA export is required for its translation into functional protein, facilitated by the interaction between mRNA transporters and nuclear pore complex (NPC). However, only a few mRNA transporters associated with NPC were identified. Here, we comprehensively depicted NPC interactome in human embryonic stem cells (hESCs), from which we identified a series of novel mRNA transporters. Among them, FXR1 functioned as a cytoplasmic mRNA acceptor, promoted the release of G-quadruplex containing mRNA from its export receptor, simultaneously mediated the localization of nucleoporins mRNA on nuclear pore. FXR1 bound G-quadruplex or nucleoporin AGAA RNA via its RGG and KH domain respectively, which in turn weakened or enhanced its binding to nuclear pore, thus resulted in mRNA release or localization. More importantly, the decline of FXR1 and nuclear pore activity helped hESCs to achieve fate transition by impeding the nuclear export of transcribed RNAs, which was required for hESC differentiation.
2025-08-22 | GSE245777 | GEO
Project description:Abnormal distribution of cellular cholesterol is associated with numerous diseases, including cardiovascular and neurodegenerative diseases. Regulated transport of cholesterol is critical for maintaining its proper distribution in the cell, yet the underlying mechanisms remain unclear. Here, we show that lipid transfer proteins, namely ORP9, OSBP, and GRAMD1s/Asters (GRAMD1a/GRAMD1b/GRAMD1c), control non-vesicular cholesterol transport at points of contact between the ER and the trans-Golgi network (TGN), thereby maintaining cellular cholesterol distribution. ORP9 localizes to the TGN via interaction between its tandem α-helices and ORP10/ORP11. ORP9 extracts PI4P from the TGN to prevent its overaccumulation and suppresses OSBP-mediated PI4P-driven cholesterol transport to the Golgi. By contrast, GRAMD1s transport excess cholesterol from the Golgi to the ER, thereby preventing its build-up. Cells lacking ORP9 exhibit accumulation of cholesterol at the Golgi, which is further enhanced by additional depletion of GRAMD1s with major accumulation in the plasma membrane. This is accompanied by chronic activation of the SREBP-2 signalling pathway. Our findings reveal the importance of regulated lipid transport at ER-Golgi contacts for maintaining cellular cholesterol distribution and homeostasis.
2023-09-24 | GSE240960 | GEO
Project description:The piRNA pathway is a conserved small RNA-based immune system that protects animal germ cell genomes from the harmful effects of transposon mobilisation. In Drosophila ovaries, most piRNAs originate from dual-strand clusters, which generate piRNAs from both genomic strands. Dual-strand clusters use non-canonical transcription mechanisms. Although transcribed by RNA polymerase II, cluster transcripts lack splicing signatures and polyA tails. mRNA processing is important for general mRNA export mediated by Nuclear export factor 1. Although UAP56, a component of the transcription and export complex, has been implicated in piRNA precursor export, it remains unknown how dual-strand cluster transcripts are specifically targeted for piRNA biogenesis by export from the nucleus to cytoplasmic processing centers. Here we report that dual-strand cluster transcript export requires CG13741/Bootlegger and the Drosophila Nuclear export factor family protein, Nxf3. Bootlegger is specifically recruited to piRNA clusters and in turn brings Nxf3. We find that Nxf3 specifically binds to piRNA precursors and is essential for their export to piRNA biogenesis sites, a process that is critical for germline transposon silencing. Our data shed light on how dual-strand clusters bypass canonical mRNA features to be specifically exported via Nxf3, ensuring proper piRNA production.
2019-07-27 | GSE133528 | GEO
Project description:Circular RNAs (circRNAs), which can function as regulators of gene expression, are formed by back-splicing of precursor mRNAs in the nucleus. circRNAs are predominantly localized in the cytoplasm, indicating that they must be exported from the nucleus. Here, we uncover a pathway specific for nuclear export of circular RNA. This pathway requires Ran-GTP, Exportin-2 and IGF2BP1. Enhancing the nuclear Ran-GTP gradient by depletion or chemical inhibition of the major protein exporter, CRM1, selectively increases nuclear export of circRNAs, while reducing the nuclear Ran-GTP gradient selectively blocks circRNA export. Analysis of nuclear circRNA binding proteins reveals that interaction of IGF2BP1 with circRNA is enhanced by Ran-GTP, whereas its interaction with linear RNA is inhibited by Ran-GTP. Depletion or knockout of Exportin-2 specifically inhibits nuclear export of circRNA, while formation of an Exportin-2 circRNA export complex requires Ran-GTP and IGF2BP1. Our findings demonstrate that adaptors such as IGF2BP1 that bind directly to circular RNAs recruit Exportin-2 to export circRNAs in a mechanism analogous to protein export, rather than mRNA export.
2023-12-09 | GSE235899 | GEO
Project description:Circular RNAs (circRNAs), which are increasingly being implicated in a variety of functions in normal and cancerous cells1-5, are formed by back-splicing of precursor mRNAs in the nucleus6-10. circRNAs are predom¬inantly localized in the cytoplasm, indicating that they must be exported from the nucleus. Here, we uncover a pathway specific for nuclear export of circular RNA. This pathway requires Ran-GTP, Exportin-2 and IGF2BP1. Enhancing the nuclear Ran-GTP gradient by depletion or chemical inhibition of the major protein exporter, CRM1, selectively increases nuclear export of circRNAs, while reducing the nuclear Ran-GTP gradient selectively blocks circRNA export. Depletion or knockout of Exportin-2 specifically inhibits nuclear export of circRNA. Analysis of nuclear circRNA binding proteins reveals that interaction of IGF2BP1 with circRNA is enhanced by Ran-GTP. Formation of circRNA export complexes in the nucleus is promoted by Ran-GTP through its interactions with IGF2BP1, Exportin-2 and circRNA. Our findings demonstrate that adaptors such as IGF2BP1 that bind directly to circular RNAs recruit Ran-GTP and Exportin-2 to export circRNAs in a mechanism analogous to protein export, rather than mRNA export.
2023-11-22 | GSE226716 | GEO