RESUMEN
Somatic mutations in the spliceosome gene U2AF1 are common in patients with myelodysplastic syndromes. U2AF1 mutations that code for the most common amino acid substitutions are always heterozygous, and the retained WT allele is expressed, suggesting that mutant hematopoietic cells may require the residual WT allele to be viable. We show that hematopoiesis and RNA splicing in U2af1 heterozygous knockout mice were similar to those in control mice, but that deletion of the WT allele in U2AF1(S34F) heterozygous mutant-expressing hematopoietic cells (i.e., hemizygous mutant) was lethal. These results confirm that U2AF1 mutant hematopoietic cells are dependent on the expression of WT U2AF1 for survival in vivo and that U2AF1 is a haplo-essential cancer gene. Mutant U2AF1(S34F)-expressing cells were also more sensitive to reduced expression of WT U2AF1 than nonmutant cells. Furthermore, mice transplanted with leukemia cells expressing mutant U2AF1 had significantly reduced tumor burden and improved survival after the WT U2af1 allele was deleted compared with when it was not deleted. These results suggest that selectively targeting the WT U2AF1 allele in heterozygous mutant cells could induce cancer cell death and be a therapeutic strategy for patients harboring U2AF1 mutations.
Asunto(s)
Alelos , Neoplasias Hematológicas , Heterocigoto , Leucemia , Proteínas de Neoplasias , Neoplasias Experimentales , Factor de Empalme U2AF , Animales , Neoplasias Hematológicas/genética , Neoplasias Hematológicas/metabolismo , Leucemia/genética , Leucemia/metabolismo , Ratones , Ratones Noqueados , Proteínas de Neoplasias/biosíntesis , Proteínas de Neoplasias/genética , Neoplasias Experimentales/genética , Neoplasias Experimentales/metabolismo , Factor de Empalme U2AF/biosíntesis , Factor de Empalme U2AF/genéticaRESUMEN
Nonsense-mediated RNA decay (NMD) is recognized as an RNA surveillance pathway that targets aberrant mRNAs with premature translation termination codons (PTC) for degradation, however, its molecular mechanisms and roles in health and disease remain incompletely understood. In this study, we developed a novel reporter system to accurately measure NMD activity in individual cells. A genome-wide CRISPR-Cas9 knockout screen using this reporter system identified novel NMD-promoting factors, including multiple components of the SF3B complex and other U2 spliceosome factors. Interestingly, cells with mutations in the spliceosome genes SF3B1 and U2AF1, which are commonly found in myelodysplastic syndrome (MDS) and cancers, have overall attenuated NMD activity. Compared with wild-type (WT) cells, SF3B1- and U2AF1-mutant cells were more sensitive to NMD inhibition, a phenotype that is accompanied by elevated DNA replication obstruction, DNA damage, and chromosomal instability. Remarkably, the sensitivity of spliceosome mutant cells to NMD inhibition was rescued by overexpression of RNase H1, which removes R-loops in the genome. Together, these findings shed new light on the functional interplay between NMD and RNA splicing and suggest a novel synthetic lethal strategy for the treatment of MDS and cancers with spliceosome mutations. SIGNIFICANCE: This study has developed a novel NMD reporter system and identified a potential therapeutic approach of targeting the NMD pathway to treat cancer with spliceosome gene mutations.
Asunto(s)
Mutación , Síndromes Mielodisplásicos/metabolismo , Degradación de ARNm Mediada por Codón sin Sentido , Fosfoproteínas/genética , Factores de Empalme de ARN/genética , Factor de Empalme U2AF/genética , Ciclo Celular , Línea Celular Tumoral , Inestabilidad Cromosómica , Colorantes Fluorescentes , Regulación de la Expresión Génica , Genes Reporteros , Estudio de Asociación del Genoma Completo , Humanos , Células K562 , Proteínas de Unión al ARN , RNA-Seq , Ribonucleasa H/metabolismo , EmpalmosomasRESUMEN
Heterozygous somatic mutations in the spliceosome gene U2AF1 occur in â¼ 11% of patients with myelodysplastic syndromes (MDS), the most common adult myeloid malignancy. It is unclear how these mutations contribute to disease. We examined in vivo hematopoietic consequences of the most common U2AF1 mutation using a doxycycline-inducible transgenic mouse model. Mice expressing mutant U2AF1(S34F) display altered hematopoiesis and changes in pre-mRNA splicing in hematopoietic progenitor cells by whole transcriptome analysis (RNA-seq). Integration with human RNA-seq datasets determined that common mutant U2AF1-induced splicing alterations are enriched in RNA processing genes, ribosomal genes, and recurrently mutated MDS and acute myeloid leukemia-associated genes. These findings support the hypothesis that mutant U2AF1 alters downstream gene isoform expression, thereby contributing to abnormal hematopoiesis in patients with MDS.
Asunto(s)
Hematopoyesis/genética , Mutación , Proteínas Nucleares/genética , Precursores del ARN/genética , Empalme del ARN/genética , ARN Mensajero/genética , Ribonucleoproteínas/genética , Animales , Humanos , Leucemia Mieloide Aguda/genética , Ratones Transgénicos , Síndromes Mielodisplásicos/genética , Factor de Empalme U2AFRESUMEN
Numerous mouse models have utilized Cre-loxP technology to modify gene expression. Adverse effects of Cre recombinase activity have been reported, including in the heart. However, the mechanisms associated with cardiac Cre toxicity are largely unknown. Here, we show that expression of Cre in cardiomyocytes induces a DNA damage response, resulting in cardiomyocyte apoptosis, cardiac fibrosis and cardiac dysfunction. In an effort to increase the recombination efficiency of a widely used tamoxifen-sensitive Cre transgene under control of the α-myosin-heavy-chain promoter (αMHC-MerCreMer), we observed myocardial dysfunction and decreased survival, which were dependent on the dose of tamoxifen injected. After excluding a Cre-independent contribution by tamoxifen, we found that Cre induced myocardial fibrosis, activation of pro-fibrotic genes and cardiomyocyte apoptosis. Examination of the molecular mechanisms showed activation of DNA damage response signaling and p53 stabilization in the absence of loxP sites, suggesting that Cre induced illegitimate DNA breaks. Cardiomyocyte apoptosis was also induced by expressing Cre using adenoviral transduction, indicating that the effect was not dependent on genomic integration of the transgene. Cre-mediated homologous recombination at loxP sites was dose-dependent and had a ceiling effect at â¼80% of cardiomyocytes showing recombination. By titrating the amount of tamoxifen to maximize recombination while minimizing animal lethality, we determined that 30 µg tamoxifen/g body weight/day injected on three consecutive days is the optimal condition for the αMHC-MerCreMer system to induce recombination in the Rosa26-lacZ strain. Our results further highlight the importance of experimental design, including the use of appropriate genetic controls for Cre expression.
Asunto(s)
Daño del ADN/efectos de los fármacos , Insuficiencia Cardíaca/inducido químicamente , Miocitos Cardíacos/efectos de los fármacos , Análisis de Supervivencia , Tamoxifeno/farmacología , Animales , Apoptosis , Relación Dosis-Respuesta a Droga , Ratones , Miocitos Cardíacos/patología , Tamoxifeno/administración & dosificación , Tamoxifeno/efectos adversosRESUMEN
The signaling complex consisting of the growth factor neuregulin-1 (NRG1) and its tyrosine kinase receptors ErbB2 and ErbB4 has a critical role in cardiac development and homeostasis of the structure and function of the adult heart. Recent research results suggest that targeting this signaling complex may provide a viable strategy for treating heart failure. Clinical trials are currently evaluating the effectiveness and safety of intravenous administration of recombinant NRG1 formulations in heart failure patients. Endogenous as well as administered NRG1 has multiple possible activities in the adult heart, but how these are related is unknown. It has recently been demonstrated that NRG1 administration can stimulate proliferation of cardiomyocytes, which may contribute to repair failing hearts. This review summarizes the current knowledge of how NRG1 and its receptors control cardiac physiology and biology, with special emphasis on its role in cardiomyocyte proliferation during myocardial growth and regeneration.
Asunto(s)
Proliferación Celular , Receptores ErbB/fisiología , Miocitos Cardíacos/fisiología , Neurregulinas/fisiología , Receptor ErbB-2/fisiología , Transducción de Señal/fisiología , Animales , Insuficiencia Cardíaca/tratamiento farmacológico , Humanos , Ratones , Modelos Animales , Miocitos Cardíacos/citología , Neurregulina-1/uso terapéutico , Neurregulinas/uso terapéutico , Ratas , Receptor ErbB-4 , Proteínas Recombinantes/uso terapéuticoRESUMEN
With the goal of elucidating the molecular and cellular mechanisms of chloroprene toxicity, we examined the potential DNA cross-linking of the bifunctional chloroprene metabolite, (1-chloroethenyl)oxirane (CEO). We used denaturing polyacrylamide gel electrophoresis to monitor the possible formation of interstrand cross-links by CEO within synthetic DNA duplexes. Our data suggest interstrand cross-linking at deoxyguanosine residues within 5'-GC and 5'-GGC sites, with the rate of cross-linking depending on pH (pH 5.0 > pH 6.0 > pH 7.0). A comparison of the cross-linking efficiencies of CEO and the structurally similar cross-linkers diepoxybutane (DEB) and epichlorohydrin (ECH) revealed that DEB > CEO > or = ECH. Furthermore, we found that cytotoxicity correlates with cross-linking efficiency, supporting a role for interstrand cross-links in the genotoxicology of chloroprene.