RESUMO
MID1/TRIM18 is a member of the TRIM family of ubiquitin E3 ligases characterized by the presence of a conserved RING-containing N-terminal tripartite motif. Mutations in the MID1 gene have been associated with the X-linked form of Opitz Syndrome, a developmental disorder characterized by midline defects and intellectual disability. The effect of MID1 E3 ligase activity within the cell and the role in the pathogenesis of the disease is still not completely unraveled. Here, we report BRAF35, a non-canonical HMG nuclear factor, as a novel MID1 substrate. MID1 is implicated in BRAF35 ubiquitination promoting atypical poly-ubiquitination via K6-, K27- and K29-linkages. We observed a partial co-localization of the two proteins within cytoplasmic bodies. We found that MID1 depletion alters BRAF35 localization in these structures and increases BRAF35 stability affecting its cytoplasmic abundance. Our data reveal a novel role for MID1 and for atypical ubiquitination in balancing BRAF35 presence, and likely its activity, within nuclear and cytoplasmic compartments.
Assuntos
Fissura Palatina/genética , Esôfago/anormalidades , Doenças Genéticas Ligadas ao Cromossomo X/genética , Proteínas de Grupo de Alta Mobilidade/genética , Hipertelorismo/genética , Hipospadia/genética , Proteínas dos Microtúbulos/genética , Proteínas Nucleares/genética , Fatores de Transcrição/genética , Ubiquitina-Proteína Ligases/genética , Sequência de Aminoácidos , Fissura Palatina/patologia , Citoplasma/enzimologia , Esôfago/patologia , Doenças Genéticas Ligadas ao Cromossomo X/patologia , Humanos , Hipertelorismo/patologia , Hipospadia/patologia , Proteínas dos Microtúbulos/metabolismo , Mutação , Proteínas Nucleares/metabolismo , Proteína Fosfatase 2/genética , Proteína Fosfatase 2/metabolismo , Fatores de Transcrição/metabolismo , Ubiquitina/genética , Ubiquitina/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , UbiquitinaçãoRESUMO
DNA replication initiates from multiple origins, and selective CDC7 kinase inhibitors (CDC7is) restrain cell proliferation by limiting origin firing. We have performed a CRISPR-Cas9 genome-wide screen to identify genes that, when lost, promote the proliferation of cells treated with sub-efficacious doses of a CDC7i. We have found that the loss of function of ETAA1, an ATR activator, and RIF1 reduce the sensitivity to CDC7is by allowing DNA synthesis to occur more efficiently, notably during late S phase. We show that partial CDC7 inhibition induces ATR mainly through ETAA1, and that if ATR is subsequently inhibited, origin firing is unleashed in a CDK- and CDC7-dependent manner. Cells are then driven into a premature and highly defective mitosis, a phenotype that can be recapitulated by ETAA1 and TOPBP1 co-depletion. This work defines how ATR mediates the effects of CDC7 inhibition, establishing the framework to understand how the origin firing checkpoint functions.