RESUMO
PRDM16 is a dynamic transcriptional regulator of various stem cell niches, including adipocytic, hematopoietic, cardiac progenitors, and neural stem cells. PRDM16 has been suggested to contribute to 1p36 deletion syndrome, one of the most prevalent subtelomeric microdeletion syndromes. We report a patient with a de novo nonsense mutation in the PRDM16 coding sequence, accompanied by lissencephaly and microcephaly features. Human stem cells were genetically modified to mimic this mutation, generating cortical organoids that exhibited altered cell cycle dynamics. RNA sequencing of cortical organoids at day 32 unveiled changes in cell adhesion and WNT-signaling pathways. ChIP-seq of PRDM16 identified binding sites in postmortem human fetal cortex, indicating the conservation of PRDM16 binding to developmental genes in mice and humans, potentially at enhancer sites. A shared motif between PRDM16 and LHX2 was identified and further examined through comparison with LHX2 ChIP-seq data from mice. These results suggested a collaborative partnership between PRDM16 and LHX2 in regulating a common set of genes and pathways in cortical radial glia cells, possibly via their synergistic involvement in cortical development.
RESUMO
The adult human brain comprises more than a thousand distinct neuronal and glial cell types, a diversity that emerges during early brain development. To reveal the precise sequence of events during early brain development, we used single-cell RNA sequencing and spatial transcriptomics and uncovered cell states and trajectories in human brains at 5 to 14 postconceptional weeks (pcw). We identified 12 major classes that are organized as ~600 distinct cell states, which map to precise spatial anatomical domains at 5 pcw. We described detailed differentiation trajectories of the human forebrain and midbrain and found a large number of region-specific glioblasts that mature into distinct pre-astrocytes and pre-oligodendrocyte precursor cells. Our findings reveal the establishment of cell types during the first trimester of human brain development.
Assuntos
Encéfalo , Neurogênese , Primeiro Trimestre da Gravidez , Feminino , Humanos , Gravidez , Astrócitos/citologia , Encéfalo/citologia , Encéfalo/embriologia , Neuroglia , Neurônios/citologia , Atlas como Assunto , Análise da Expressão Gênica de Célula ÚnicaRESUMO
PRDM16 is a dynamic transcriptional regulator of various stem cell niches, including adipocytic, hematopoietic, cardiac progenitors, and neural stem cells. PRDM16 has been suggested to contribute to 1p36 deletion syndrome, one of the most prevalent subtelomeric microdeletion syndromes. We report a patient with a de novo nonsense mutation in the PRDM16 coding sequence, accompanied by lissencephaly and microcephaly features. Human stem cells were genetically modified to mimic this mutation, generating cortical organoids that exhibited altered cell cycle dynamics. RNA sequencing of cortical organoids at day 32 unveiled changes in cell adhesion and WNT-signaling pathways. ChIP-seq of PRDM16 identified binding sites in postmortem human fetal cortex, indicating the conservation of PRDM16 binding to developmental genes in mice and humans, potentially at enhancer sites. A shared motif between PRDM16 and LHX2 was identified and further examined through comparison with LHX2 ChIP-seq data from mice. These results suggested a collaborative partnership between PRDM16 and LHX2 in regulating a common set of genes and pathways in cortical radial glia cells, possibly via their synergistic involvement in cortical development.
RESUMO
Pancreatic ductal adenocarcinoma (PDA) is characterized by aggressive local invasion and metastatic spread, leading to high lethality. Although driver gene mutations during PDA progression are conserved, no specific mutation is correlated with the dissemination of metastases1-3. Here we analysed RNA splicing data of a large cohort of primary and metastatic PDA tumours to identify differentially spliced events that correlate with PDA progression. De novo motif analysis of these events detected enrichment of motifs with high similarity to the RBFOX2 motif. Overexpression of RBFOX2 in a patient-derived xenograft (PDX) metastatic PDA cell line drastically reduced the metastatic potential of these cells in vitro and in vivo, whereas depletion of RBFOX2 in primary pancreatic tumour cell lines increased the metastatic potential of these cells. These findings support the role of RBFOX2 as a potent metastatic suppressor in PDA. RNA-sequencing and splicing analysis of RBFOX2 target genes revealed enrichment of genes in the RHO GTPase pathways, suggesting a role of RBFOX2 splicing activity in cytoskeletal organization and focal adhesion formation. Modulation of RBFOX2-regulated splicing events, such as via myosin phosphatase RHO-interacting protein (MPRIP), is associated with PDA metastases, altered cytoskeletal organization and the induction of focal adhesion formation. Our results implicate the splicing-regulatory function of RBFOX2 as a tumour suppressor in PDA and suggest a therapeutic approach for metastatic PDA.
Assuntos
Processamento Alternativo , Carcinoma Ductal Pancreático , Neoplasias Pancreáticas , Humanos , Processamento Alternativo/genética , Carcinoma Ductal Pancreático/genética , Carcinoma Ductal Pancreático/patologia , Linhagem Celular Tumoral , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/patologia , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Fatores de Processamento de RNA/genética , Fatores de Processamento de RNA/metabolismo , Animais , Metástase Neoplásica , Adesões FocaisRESUMO
Alternative splicing, a fundamental step in gene expression, is deregulated in many diseases. Splicing factors (SFs), which regulate this process, are up- or down regulated or mutated in several diseases including cancer. To date, there are no inhibitors that directly inhibit the activity of SFs. We designed decoy oligonucleotides, composed of several repeats of a RNA motif, which is recognized by a single SF. Here we show that decoy oligonucleotides targeting splicing factors RBFOX1/2, SRSF1 and PTBP1, can specifically bind to their respective SFs and inhibit their splicing and biological activities both in vitro and in vivo. These decoy oligonucleotides present an approach to specifically downregulate SF activity in conditions where SFs are either up-regulated or hyperactive.
Assuntos
Ribonucleoproteínas Nucleares Heterogêneas/genética , Oligonucleotídeos/farmacologia , Proteína de Ligação a Regiões Ricas em Polipirimidinas/genética , Fatores de Processamento de RNA/genética , Fatores de Processamento de Serina-Arginina/genética , Processamento Alternativo , Animais , Animais Geneticamente Modificados , Sítios de Ligação , Glioblastoma/genética , Glioblastoma/patologia , Células HEK293 , Ribonucleoproteínas Nucleares Heterogêneas/antagonistas & inibidores , Ribonucleoproteínas Nucleares Heterogêneas/metabolismo , Humanos , Sistema de Sinalização das MAP Quinases/genética , Músculo Esquelético/crescimento & desenvolvimento , Degradação do RNAm Mediada por Códon sem Sentido , Oligonucleotídeos/química , Oligonucleotídeos/metabolismo , Proteína de Ligação a Regiões Ricas em Polipirimidinas/antagonistas & inibidores , Proteína de Ligação a Regiões Ricas em Polipirimidinas/metabolismo , Fatores de Processamento de RNA/antagonistas & inibidores , Fatores de Processamento de RNA/metabolismo , Fatores de Processamento de Serina-Arginina/antagonistas & inibidores , Fatores de Processamento de Serina-Arginina/metabolismo , Sequências de Repetição em Tandem , Ensaios Antitumorais Modelo de Xenoenxerto , Peixe-Zebra/embriologia , Peixe-Zebra/genéticaRESUMO
RNA-editing mechanisms, which induce nucleotide substitution in the RNA, increase transcript and protein diversities. Editing dysregulation has been shown to lead to grave outcomes, and transcriptome-wide aberrant RNA editing has been found in tumors. However, little is known about the involvement of editing in other diseases. Systemic lupus erythematosus (SLE) is a multisystemic autoimmune disease characterized by a loss of tolerance for autoantigens from various tissues and the production of multiple autoantibodies. Here, we show that blood samples from individuals with SLE have abnormally high levels of RNA editing, some of which affect proteins and potentially generate novel autoantigens. We suggest that elevated RNA editing, either by ADARs or APOBECs, may be involved in the pathophysiology of SLE, as well as in other autoimmune diseases, by generating or increasing the autoantigen load, a key requisite for the progression of autoimmunity.
Assuntos
Autoantígenos/genética , Lúpus Eritematoso Sistêmico/genética , Edição de RNA , Desaminases APOBEC , Adenosina Desaminase/metabolismo , Citidina Desaminase , Citosina Desaminase/metabolismo , Humanos , Tolerância Imunológica/genética , Lúpus Eritematoso Sistêmico/imunologia , Proteínas de Ligação a RNA/metabolismoRESUMO
Concentration is important and not only while driving; a new study indicates how an adjacent genomic element helps to increase the efficiency of a specific adenosine to inosine RNA editing reaction, by providing a means to increase the local concentration of the RNA editing enzyme ADAR.
Assuntos
Inosina , Edição de RNA , Adenosina , Adenosina Desaminase/genética , Animais , TranscriptomaRESUMO
BACKGROUND: In order to become functionally competent but harmless mediators of the immune system, T cells undergo a strict educational program in the thymus, where they learn to discriminate between self and non-self. This educational program is, to a large extent, mediated by medullary thymic epithelial cells that have a unique capacity to express, and subsequently present, a large fraction of body antigens. While the scope of promiscuously expressed genes by medullary thymic epithelial cells is well-established, relatively little is known about the expression of variants that are generated by co-transcriptional and post-transcriptional processes. RESULTS: Our study reveals that in comparison to other cell types, medullary thymic epithelial cells display significantly higher levels of alternative splicing, as well as A-to-I and C-to-U RNA editing, which thereby further expand the diversity of their self-antigen repertoire. Interestingly, Aire, the key mediator of promiscuous gene expression in these cells, plays a limited role in the regulation of these transcriptional processes. CONCLUSIONS: Our results highlight RNA processing as another layer by which the immune system assures a comprehensive self-representation in the thymus which is required for the establishment of self-tolerance and prevention of autoimmunity.
Assuntos
Células Epiteliais/imunologia , Edição de RNA/genética , Timo/imunologia , Processamento Alternativo/genética , Processamento Alternativo/imunologia , Animais , Autoantígenos/genética , Autoantígenos/imunologia , Diferenciação Celular/imunologia , Regulação da Expressão Gênica/imunologia , Humanos , Camundongos , Edição de RNA/imunologia , Tolerância a Antígenos Próprios/imunologia , Linfócitos T/imunologia , Fatores de Transcrição/genéticaRESUMO
Genomic mutations in key genes are known to drive tumorigenesis and have been the focus of much attention in recent years. However, genetic content also may change farther downstream. RNA editing alters the mRNA sequence from its genomic blueprint in a dynamic and flexible way. A few isolated cases of editing alterations in cancer have been reported previously. Here, we provide a transcriptome-wide characterization of RNA editing across hundreds of cancer samples from multiple cancer tissues, and we show that A-to-I editing and the enzymes mediating this modification are significantly altered, usually elevated, in most cancer types. Increased editing activity is found to be associated with patient survival. As is the case with somatic mutations in DNA, most of these newly introduced RNA mutations are likely passengers, but a few may serve as drivers that may be novel candidates for therapeutic and diagnostic purposes.
Assuntos
Neoplasias da Mama/genética , Carcinoma/genética , Regulação Neoplásica da Expressão Gênica , Neoplasias Renais/genética , Neoplasias da Próstata/genética , Edição de RNA , Transcriptoma , Feminino , Humanos , Masculino , Mutação , Regulação para CimaRESUMO
Cancer is a complex disease that involves aberrant gene expression regulation. Discriminating the modified expression patterns driving tumor biology from the many that have no or little contribution is important for understanding cancer molecular basis. Recurrent deregulation patterns observed in multiple cancer types are enriched for such driver events. Here, we studied splicing alterations in hundreds of matched tumor and normal RNA-seq samples of eight solid cancer types. We found hundreds of cassette exons for which splicing was altered in multiple cancer types and identified a set of highly frequent altered splicing events. Specific splicing regulators, including RBFOX2, MBNL1/2 and QKI, appear to account for many splicing alteration events in multiple cancer types. Together, our results provide a first global analysis of regulated splicing alterations in cancer and identify common events with a potential causative role in solid tumor development.