RESUMO
Circular RNAs are an endogenous long-lived and abundant noncoding species. Despite their prevalence, only a few circRNAs have been dissected mechanistically to date. Here, we cataloged nascent RNA-enriched circRNAs from primary human cells and functionally assigned a role to circRAB3IP in sustaining cellular homeostasis. We combined "omics" and functional experiments to show how circRAB3IP depletion deregulates hundreds of genes, suppresses cell cycle progression, and induces senescence-associated gene expression changes. Conversely, excess circRAB3IP delivered to endothelial cells via extracellular vesicles suffices for accelerating their division. We attribute these effects to an interplay between circRAB3IP and the general splicing factor SF3B1, which can affect transcript variant expression levels of cell cycle-related genes. Together, our findings link the maintenance of cell homeostasis to the presence of a single circRNA.
Assuntos
MicroRNAs , RNA Circular , Humanos , RNA Circular/genética , Células Endoteliais/metabolismo , Proliferação de Células/genética , RNA Mensageiro/genética , Expressão Gênica , MicroRNAs/genéticaRESUMO
Glioblastoma multiforme (GBM) encompasses brain malignancies marked by phenotypic and transcriptional heterogeneity thought to render these tumors aggressive, resistant to therapy, and inevitably recurrent. However, little is known about how the spatial organization of GBM genomes underlies this heterogeneity and its effects. Here, we compile a cohort of 28 patient-derived glioblastoma stem cell-like lines (GSCs) known to reflect the properties of their tumor-of-origin; six of these were primary-relapse tumor pairs from the same patient. We generate and analyze 5 kbp-resolution chromosome conformation capture (Hi-C) data from all GSCs to systematically map thousands of standalone and complex structural variants (SVs) and the multitude of neoloops arising as a result. By combining Hi-C, histone modification, and gene expression data with chromatin folding simulations, we explain how the pervasive, uneven, and idiosyncratic occurrence of neoloops sustains tumor-specific transcriptional programs via the formation of new enhancer-promoter contacts. We also show how even moderately recurrent neoloops can relate to patient-specific vulnerabilities. Together, our data provide a resource for dissecting GBM biology and heterogeneity, as well as for informing therapeutic approaches.
Assuntos
Neoplasias Encefálicas , Cromatina , Regulação Neoplásica da Expressão Gênica , Glioblastoma , Glioblastoma/genética , Glioblastoma/patologia , Humanos , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/patologia , Cromatina/metabolismo , Cromatina/genética , Células-Tronco Neoplásicas/metabolismo , Células-Tronco Neoplásicas/patologia , Linhagem Celular Tumoral , Heterogeneidade Genética , Regiões Promotoras Genéticas/genética , Transcrição Gênica , Elementos Facilitadores Genéticos/genética , Cromossomos Humanos/genéticaRESUMO
Cellular senescence is acknowledged as a key contributor to organismal ageing and late-life disease. Though popular, the study of senescence in vitro can be complicated by the prolonged and asynchronous timing of cells committing to it and by its paracrine effects. To address these issues, we repurposed a small molecule inhibitor, inflachromene (ICM), to induce senescence to human primary cells. Within 6 days of treatment with ICM, senescence hallmarks, including the nuclear eviction of HMGB1 and -B2, are uniformly induced across IMR90 cell populations. By generating and comparing various high throughput datasets from ICM-induced and replicative senescence, we uncovered a high similarity of the two states. Notably though, ICM suppresses the pro-inflammatory secretome associated with senescence, thus alleviating most paracrine effects. In summary, ICM rapidly and synchronously induces a senescent-like phenotype thereby allowing the study of its core regulatory program without confounding heterogeneity.
Assuntos
Envelhecimento , Senescência Celular , Humanos , Envelhecimento/genética , Senescência Celular/genéticaRESUMO
Large-scale genomic profiling of pancreatic cancer (PDAC) has revealed two distinct subtypes: 'classical' and 'basal-like'. Their variable coexistence within the stromal immune microenvironment is linked to differential prognosis; however, the extent to which these neoplastic subtypes shape the stromal immune landscape and impact clinical outcome remains unclear. By combining preclinical models, patient-derived xenografts, as well as FACS-sorted PDAC patient biopsies, we show that the basal-like neoplastic state is sustained via BRD4-mediated cJUN/AP1 expression, which induces CCL2 to recruit tumor necrosis factor (TNF)-α-secreting macrophages. TNF-α+ macrophages force classical neoplastic cells into an aggressive phenotypic state via lineage reprogramming. Integration of ATAC-, ChIP- and RNA-seq data revealed distinct JUNB/AP1 (classical) and cJUN/AP1 (basal-like)-driven regulation of PDAC subtype identity. Pharmacological inhibition of BRD4 led to suppression of the BRD4-cJUN-CCL2-TNF-α axis, restoration of classical subtype identity and a favorable prognosis. Hence, patient-tailored therapy for a cJUNhigh/TNF-αhigh subtype is paramount in overcoming highly inflamed and aggressive PDAC states.
Assuntos
Carcinoma Ductal Pancreático , Neoplasias Pancreáticas , Carcinoma Ductal Pancreático/genética , Proteínas de Ciclo Celular/genética , Regulação Neoplásica da Expressão Gênica , Humanos , Macrófagos/metabolismo , Proteínas Nucleares/genética , Neoplasias Pancreáticas/genética , Prognóstico , Fatores de Transcrição/genética , Microambiente Tumoral/genética , Fator de Necrose Tumoral alfa/genética , Neoplasias PancreáticasRESUMO
Sensitive factor attachment protein receptors (SNARE) proteins are important mediators of protein trafficking that regulate the membrane fusion of specific vesicle populations and their target organelles. The SNARE protein Ykt6 lacks a transmembrane domain and attaches to different organelle membranes. Mechanistically, Ykt6 activity is thought to be regulated by a conformational change from a closed cytosolic form to an open membrane-bound form, yet the mechanism that regulates this transition is unknown. We identified phosphorylation sites in the SNARE domain of Ykt6 that mediate Ykt6 membrane recruitment and are essential for cellular growth. Using proximity-dependent labeling and membrane fractionation, we found that phosphorylation regulates Ykt6 conversion from a closed to an open conformation. This conformational switch recruits Ykt6 to several organelle membranes, where it functionally regulates the trafficking of Wnt proteins and extracellular vesicle secretion in a concentration-dependent manner. We propose that phosphorylation of its SNARE domain leads to a conformational switch from a cytosolic, auto-inhibited Ykt6 to an active SNARE at different membranes.