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Locolization of circRNA with DIS3 WT or D146N mutant in sucrose density gradient centrifugation assay

ABSTRACT: Locolization of circRNA with DIS3 WT or D146N mutant in sucrose density gradient centrifugation assay

PROVIDER: PRJNA1079793 | ENA |

REPOSITORIES: ENA

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Locolization of circRNA with DIS3 WT or D146N mutant in sucrose density gradient centrifugation assay

Project description:To investigate the locolization between DIS3 and circRNA in fractions form sucrose density gradient centrifugation (SDGC) assay for 293T cells

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Project description:his dataset examines the proteomic remodeling of phase-separated protein condensates in response to high-magnesium stimulation, utilizing discontinuous sucrose density gradient ultracentrifugation combined with ultra-low input mass spectrometry. RIPA cell lysates were layered onto a stepwise sucrose gradient (10%, 22.5%, and 35% from bottom to top) and centrifuged at 250,000 g for 4 hours at 4°C. Three fractions (top, middle, and bottom 35% dense layer) were collected post-centrifugation. To identify putative phase-separation candidates, we compared protein distribution in every density bottom fraction between human PASMC cells with low endogenous UBXN1 expression and human cells overexpressing UBXN1 under high-magnesium treatment (24–48 h).

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Depletion of DIS3 from human cell lines

Project description:DIS3 is a 3’ to 5’ exoribonuclease responsible for degradation of multiple RNA targets. Most reports in human cells focus on its function in RNA surveillance in the nucleus. However, a small fraction of DIS3 can be also found in cytoplasm. Thus, the focus of this experiment was to identify the cytoplasmic targets of DIS3. Using rapid, auxin inducible, degradation of DIS3 protein and subcellular fractionation, we were able to identify RNA accumulated in cytoplasm of human cells upon DIS3 depletion. The raw data and counts for annotated and non-annotated transcripts resulting from this experiment are provided.

2026-06-02 | GSE188195 | GEO

Ribosome profiling on polysomes extracted via sucrose gradient and IP

Project description:Technical feasibility study to determine data quality and reproducibilty. Ribosome profiling was performed by either sucrose gradient (SG) or immunoprecipitation (IP).

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Project description:CUT&Tag was performed to identify the effect of DIS3 depletion on DNA:RNA hybrids genome-wide. DIS3-AID HCT116 cells were treated with either Auxin (for rapid depletion of DIS3) or DMSO (control). CUT&Tag was performed targeting DNA:RNA hybrids using the CUT&Tag-IT® R-loop Assay Kit (Active Motive) with the S9.6 antibody.

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Ribosome profiling on polysomes extracted via sucrose gradient and IP

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Oncogenic mechanisms of DIS3 mutations in Multiple Myeloma

Project description:DIS3 is mutated in multiple myeloma (MM), but the mechanism by which oncogenesis occurs specifically in B cell lineage remains to be established. Somatic DIS3 variants have substitutions enriched around its RNB domain, which in a dominant negative way, inactivate or reduce the exoribonucleolytic activity of this enzyme responsible for nuclear RNA degradation. Here using knock-in mice with a clinical Dis3 G766R variant, we demonstrate a B cell-specific mutagenic effect that induces aberrant chromosomal translocations, increasing the incidence of plasmacytoma, a mouse model of early-stage MM. Dis3 G766R -dependent translocations display characteristics typical to aberrant activation-induced deaminase (AID) activity sites. Indeed, analysis of MM clinical samples revealed that in MM driver genes, DIS3 alleles lead to increased AID-dependent DNA lesions. Mechanistically, mutated DIS3 accumulates on chromatin-associated RNA substrates, including aberrant AID action sites, fostering oncogenic chromosomal rearrangements. Translocations occur during immunoglobulin class switch recombination, which otherwise proceeds unaffected both MM and the mouse model. In conclusion, MM DIS3 mutations lead to a gain-of-function phenotype and drive MM development, enhancing driver translocations.

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