Project description:To investigate the locolization between DIS3 and circRNA in fractions form sucrose density gradient centrifugation (SDGC) assay for 293T cells
Project description:We investigated the effect on miRNA expression in Drosophila melanogaster wing imaginal discs following the knockdown of the 3'-5' exoribonuclease Dis3.
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.
Project description:CUT&Tag was performed to identify the effect of DIS3 depletion on RNAPII in response to UV irradiation. DIS3-AID HCT116 cells were treated with either Auxin (for rapid depletion of DIS3) or DMSO (control) before irradiation with 8 J/m2 UVC. CUT&Tag was performed targeting RNAPII-Ser2p or RNAPII-Ser5p.
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.
Project description:Somatic mutations affecting DIS3, the catalytic component of the RNA exosome, have been found in up to 18% of patients affected by the hematological cancer multiple myeloma. Here we show that DIS3 targets and degrades the pluripotency factor LIN28B. In cancer cells, DIS3 inactivation leads to enhanced LIN28B expression, thus disrupting the let-7 miRNAs tumor suppressor network and ultimately increasing protein levels of crucial oncogenes such as MYC and RAS. DIS3 represents the catalytic component of the exosome. The exosome is required for cell viability and targets several RNA species, including pre-mRNAs, pre-tRNAs, pre-rRNAs, snRNAs and snoRNAs. To gain insight on the macular wiring underlying DIS3 activity in mammalian cells, we comprehensively evaluated expression profiles, including miRNAs, in various cell lines, upon DIS3 knockdown. This series of microarray experiments contains the miRNA expression profiles of independent replicates of RPMI-8226, KMS12-BM multiple myeloma cell lines and HEK-293T cells, knocked-down with a scrambled or hDIS3 sh4 and collected 72 hours after infection. 500 nanograms of total RNA were processed using the FlashTag labeling kit, which uses a tailing reaction followed by ligation of the biotinylated signal molecule to the target RNA sample. The labelled RNA was then hybridized to Affymetrix GeneChip® microRNA arrays v1.0, following the Affymetrix manufacturer's instructions.
Project description:It is well established that exposure to UV light alters the distribution of RNA polymerase II (RNAPII), but the impact of UV irradiation on the genomic distribution of DIS3 has not been investigated. We generated ChIP-seq profiles of DIS3 in U2OS cells that express a HA-tagged DIS3 protein (DIS3FLAGHA). We analyzed the total occupancy of RNAPII using an antibody against the N-terminal domain of RPB1 (NTD). Average RNAPII occupancy at transcription start sites (TSSs) was significantly decrease 3 hours after UV irradiation, consistent with previous reports, and was notably recovered 8 hours after irradiation. Upon UV irradiation, DIS3 exhibited a redistribution that closely aligned with the changes observed in RNAPII. This similarity, was particularly pronounced in highly expressed genes. Our results suggests that DIS3 is associated with the transcription machinery and can act locally at the transcription site during elongation.
