RESUMEN
Transcription factors (TFs) are important mediators of aberrant transcriptional programs in cancer cells. In this study, we focus on TF activity (TFa) as a biomarker for cell-line-selective anti-proliferative effects, in that high TFa predicts sensitivity to loss of function of a given gene (i.e., genetic dependencies [GDs]). Our linear-regression-based framework identifies 3,047 pan-cancer and 3,952 cancer-type-specific candidate TFa-GD associations from cell line data, which are then cross-examined for impact on survival in patient cohorts. One of the most prominent biomarkers is TEAD1 activity, whose associations with its predicted GDs are validated through experimental evidence as proof of concept. Overall, these TFa-GD associations represent an attractive resource for identifying innovative, biomarker-driven hypotheses for drug discovery programs in oncology.
Asunto(s)
Neoplasias , Factores de Transcripción , Humanos , Factores de Transcripción/metabolismo , Factores de Transcripción/genética , Neoplasias/genética , Neoplasias/metabolismo , Neoplasias/patología , Línea Celular Tumoral , Factores de Transcripción de Dominio TEA/metabolismo , Regulación Neoplásica de la Expresión Génica , Proteínas de Unión al ADN/metabolismo , Proteínas de Unión al ADN/genética , Biomarcadores de Tumor/metabolismo , Biomarcadores de Tumor/genética , Proliferación CelularRESUMEN
Clinical effectiveness of KRAS G12C inhibitors (G12Cis) is limited both by intrinsic and acquired resistance, necessitating the development of combination approaches. We found that targeting proximal receptor tyrosine kinase (RTK) signaling using the SOS1 inhibitor (SOS1i) BI-3406 both enhanced the potency of and delayed resistance to G12Ci treatment, but the extent of SOS1i effectiveness was modulated by both SOS2 expression and the specific mutational landscape. SOS1i enhanced the efficacy of G12Ci and limited rebound RTK/ERK signaling to overcome intrinsic/adaptive resistance, but this effect was modulated by SOS2 protein levels. Survival of drug-tolerant persister (DTP) cells within the heterogeneous tumor population and/or acquired mutations that reactivate RTK/RAS signaling can lead to outgrowth of tumor initiating cells (TICs) that drive therapeutic resistance. G12Ci drug tolerant persister cells showed a 2-3-fold enrichment of TICs, suggesting that these could be a sanctuary population of G12Ci resistant cells. SOS1i re-sensitized DTPs to G12Ci and inhibited G12C-induced TIC enrichment. Co-mutation of the tumor suppressor KEAP1 limits the clinical effectiveness of G12Cis, and KEAP1 and STK11 deletion increased TIC frequency and accelerated the development of acquired resistance to G12Ci in situ. SOS1i both delayed acquired G12Ci resistance and limited the total number of resistant colonies regardless of KEAP1 and STK11 mutational status. These data suggest that SOS1i could be an effective strategy to both enhance G12Ci efficacy and prevent G12Ci resistance regardless of co-mutations.
RESUMEN
Cohesin-mediated loop extrusion has been shown to be blocked at specific cis-elements, including CTCF sites, producing patterns of loops and domain boundaries along chromosomes. Here we explore such cis-elements, and their role in gene regulation. We find that transcription termination sites of active genes form cohesin- and RNA polymerase II-dependent domain boundaries that do not accumulate cohesin. At these sites, cohesin is first stalled and then rapidly unloaded. Start sites of transcriptionally active genes form cohesin-bound boundaries, as shown before, but are cohesin-independent. Together with cohesin loading, possibly at enhancers, these sites create a pattern of cohesin traffic that guides enhancer-promoter interactions. Disrupting this traffic pattern, by removing CTCF, renders cells sensitive to knockout of genes involved in transcription initiation, such as the SAGA complexes, and RNA processing such DEAD/H-Box RNA helicases. Without CTCF, these factors are less efficiently recruited to active promoters.
Asunto(s)
Cromatina , Proteínas Cromosómicas no Histona , Factor de Unión a CCCTC/genética , Proteínas Cromosómicas no Histona/metabolismo , Proteínas de Ciclo Celular/metabolismo , CohesinasRESUMEN
Targeted protein degradation offers an alternative modality to classical inhibition and holds the promise of addressing previously undruggable targets to provide novel therapeutic options for patients. Heterobifunctional molecules co-recruit a target protein and an E3 ligase, resulting in ubiquitylation and proteosome-dependent degradation of the target. In the clinic, the oral route of administration is the option of choice but has only been achieved so far by CRBN- recruiting bifunctional degrader molecules. We aimed to achieve orally bioavailable molecules that selectively degrade the BAF Chromatin Remodelling complex ATPase SMARCA2 over its closely related paralogue SMARCA4, to allow in vivo evaluation of the synthetic lethality concept of SMARCA2 dependency in SMARCA4-deficient cancers. Here we outline structure- and property-guided approaches that led to orally bioavailable VHL-recruiting degraders. Our tool compound, ACBI2, shows selective degradation of SMARCA2 over SMARCA4 in ex vivo human whole blood assays and in vivo efficacy in SMARCA4-deficient cancer models. This study demonstrates the feasibility for broadening the E3 ligase and physicochemical space that can be utilised for achieving oral efficacy with bifunctional molecules.
Asunto(s)
Adenosina Trifosfatasas , Factores de Transcripción , Adenosina Trifosfatasas/genética , Adenosina Trifosfatasas/metabolismo , ADN Helicasas/genética , ADN Helicasas/metabolismo , Humanos , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Proteolisis , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Ubiquitina-Proteína Ligasas/genética , Ubiquitina-Proteína Ligasas/metabolismo , Proteína Supresora de Tumores del Síndrome de Von Hippel-Lindau/genética , Proteína Supresora de Tumores del Síndrome de Von Hippel-Lindau/metabolismoRESUMEN
Genetic networks are characterized by extensive buffering. During tumor evolution, disruption of functional redundancies can create de novo vulnerabilities that are specific to cancer cells. Here, we systematically search for cancer-relevant paralog interactions using CRISPR screens and publicly available loss-of-function datasets. Our analysis reveals >2,000 candidate dependencies, several of which we validate experimentally, including CSTF2-CSTF2T, DNAJC15-DNAJC19, FAM50A-FAM50B, and RPP25-RPP25L. We provide evidence that RPP25L can physically and functionally compensate for the absence of RPP25 as a member of the RNase P/MRP complexes in tRNA processing. Our analysis also reveals unexpected redundancies between sex chromosome genes. We show that chrX- and chrY-encoded paralogs, such as ZFX-ZFY, DDX3X-DDX3Y, and EIF1AX-EIF1AY, are functionally linked. Tumor cell lines from male patients with loss of chromosome Y become dependent on the chrX-encoded gene. We propose targeting of chrX-encoded paralogs as a general therapeutic strategy for human tumors that have lost the Y chromosome.
Asunto(s)
Neoplasias , Oncogenes , ARN Helicasas DEAD-box/metabolismo , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Humanos , Masculino , Antígenos de Histocompatibilidad Menor/metabolismo , Neoplasias/genética , Proteínas de Unión al ARN/genética , Cromosomas Sexuales/metabolismo , Cromosoma X , Cromosoma YRESUMEN
Human pluripotent stem cells (hPSCs) have the capacity for self-renewal and differentiation into most cell types and, in contrast to widely used cell lines, are karyotypically normal and non-transformed. Hence, hPSCs are considered the gold-standard system for modelling diseases, especially in the field of regenerative medicine. Despite widespread research use of hPSCs and induced pluripotent stem cells (iPSCs), the systematic understanding of pluripotency and lineage differentiation mechanisms are still incomplete. Before tackling the complexities of lineage differentiation with genetic screens, it is critical to catalogue the general genetic requirements for cell fitness and proliferation in the pluripotent state and assess their plasticity under commonly used culture conditions.We describe a method to map essential genetic determinants of hPSC fitness and pluripotency, herein defined as cell reproduction, by genome-scale loss-of-function CRISPR screens in an inducible S. pyogenes Cas9 H1 hPSC line. To address questions of context-dependent gene essentiality, we include protocols for screening hPSCs cultured on feeder cells and laminin, two commonly used growth substrates. This method establishes parameters for genome-wide screens in hPSCs, making human stem cells amenable for functional genomics approaches to facilitate investigation of hPSC biology.
Asunto(s)
Células Madre Pluripotentes , Sistemas CRISPR-Cas/genética , Diferenciación Celular/genética , Células Nutrientes , Genes Esenciales , HumanosRESUMEN
We introduce Digital microfluidic Isolation of Single Cells for -Omics (DISCO), a platform that allows users to select particular cells of interest from a limited initial sample size and connects single-cell sequencing data to their immunofluorescence-based phenotypes. Specifically, DISCO combines digital microfluidics, laser cell lysis, and artificial intelligence-driven image processing to collect the contents of single cells from heterogeneous populations, followed by analysis of single-cell genomes and transcriptomes by next-generation sequencing, and proteomes by nanoflow liquid chromatography and tandem mass spectrometry. The results described herein confirm the utility of DISCO for sequencing at levels that are equivalent to or enhanced relative to the state of the art, capable of identifying features at the level of single nucleotide variations. The unique levels of selectivity, context, and accountability of DISCO suggest potential utility for deep analysis of any rare cell population with contextual dependencies.
Asunto(s)
Separación Celular/instrumentación , Análisis de la Célula Individual/instrumentación , Análisis de la Célula Individual/métodos , Animales , Antígeno CD47/genética , Línea Celular Tumoral , Separación Celular/métodos , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas , Perfilación de la Expresión Génica/instrumentación , Perfilación de la Expresión Génica/métodos , Glioblastoma/genética , Glioblastoma/metabolismo , Glioblastoma/patología , Humanos , Dispositivos Laboratorio en un Chip , Melanoma/genética , Melanoma/metabolismo , Melanoma/patología , Ratones , Técnicas Analíticas Microfluídicas/instrumentación , Técnicas Analíticas Microfluídicas/métodos , Redes Neurales de la Computación , Proteómica/métodosRESUMEN
Genome-scale functional genetic screens are used to identify key genetic regulators of a phenotype of interest. However, the identification of genetic modifications that lead to a phenotypic change requires sorting large numbers of cells, which increases operational times and costs and limits cell viability. Here, we introduce immunomagnetic cell sorting facilitated by a microfluidic chip as a rapid and scalable high-throughput method for loss-of-function phenotypic screening using CRISPR-Cas9. We used the method to process an entire genome-wide screen containing more than 108 cells in less than 1 h-considerably surpassing the throughput achieved by fluorescence-activated cell sorting, the gold-standard technique for phenotypic cell sorting-while maintaining high levels of cell viability. We identified modulators of the display of CD47, which is a negative regulator of phagocytosis and an important cell-surface target for immuno-oncology drugs. The top hit of the screen, the glutaminyl cyclase QPCTL, was validated and shown to modify the N-terminal glutamine of CD47. The method presented could bridge the gap between fluorescence-activated cell sorting and less flexible yet higher-throughput systems such as magnetic-activated cell sorting.
Asunto(s)
Genoma , Ensayos Analíticos de Alto Rendimiento/métodos , Separación Inmunomagnética/métodos , Fenotipo , Antígeno CD47/metabolismo , Sistemas CRISPR-Cas , Línea Celular Tumoral , Citometría de Flujo , Edición Génica , Humanos , Inmunoterapia , Dispositivos Laboratorio en un Chip , Neoplasias/terapiaRESUMEN
A recent study by Haapaniemi et al (2018) reported that intact p53 signaling hampers CRISPR-based functional genomic screens. Brown et al report good performance of genome-scale screens in TP53 wild-type cells and reiterate best practices for CRISPR screening.
Asunto(s)
Proteína 9 Asociada a CRISPR/genética , Sistemas CRISPR-Cas , Células Epiteliales/metabolismo , Edición Génica/normas , ARN Guía de Kinetoplastida/genética , Proteína p53 Supresora de Tumor/genética , Proteína 9 Asociada a CRISPR/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Línea Celular , Línea Celular Transformada , Línea Celular Tumoral , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/genética , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/metabolismo , Células Epiteliales/citología , Edición Génica/métodos , Regulación de la Expresión Génica , Células HCT116 , Células HeLa , Humanos , Neuroglía/metabolismo , Neuroglía/patología , Especificidad de Órganos , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Proto-Oncogénicas c-mdm2/genética , Proteínas Proto-Oncogénicas c-mdm2/metabolismo , Control de Calidad , ARN Guía de Kinetoplastida/metabolismo , Epitelio Pigmentado de la Retina/citología , Epitelio Pigmentado de la Retina/metabolismo , Proteínas de Unión a Retinoblastoma/genética , Proteínas de Unión a Retinoblastoma/metabolismo , Proteína p53 Supresora de Tumor/metabolismo , Ubiquitina-Proteína Ligasas/genética , Ubiquitina-Proteína Ligasas/metabolismoRESUMEN
The genotype-to-phenotype relationship in health and disease is complex and influenced by both an individual's environment and their unique genome. Personal genetic variants can modulate gene function to generate a phenotype either through a single gene effect or through genetic interactions involving two or more genes. The relevance of genetic interactions to disease phenotypes has been particularly clear in cancer research, where an extreme genetic interaction, synthetic lethality, has been exploited as a therapeutic strategy. The obvious benefits of unmasking genetic background-specific vulnerabilities, coupled with the power of systematic genome editing, have fueled efforts to translate genetic interaction mapping from model organisms to human cells. Here, we review recent developments in genetic interaction mapping, with a focus on CRISPR-based genome editing technologies and cancer.
Asunto(s)
Epistasis Genética , Redes Reguladoras de Genes/genética , Estudios de Asociación Genética , Neoplasias/genética , Sistemas CRISPR-Cas/genética , Edición Génica , Genotipo , Humanos , FenotipoRESUMEN
Human pluripotent stem cells (hPSCs) provide an invaluable tool for modeling diseases and hold promise for regenerative medicine. For understanding pluripotency and lineage differentiation mechanisms, a critical first step involves systematically cataloging essential genes (EGs) that are indispensable for hPSC fitness, defined as cell reproduction in this study. To map essential genetic determinants of hPSC fitness, we performed genome-scale loss-of-function screens in an inducible Cas9 H1 hPSC line cultured on feeder cells and laminin to identify EGs. Among these, we found FOXH1 and VENTX, genes that encode transcription factors previously implicated in stem cell biology, as well as an uncharacterized gene, C22orf43/DRICH1. hPSC EGs are substantially different from other human model cell lines, and EGs in hPSCs are highly context dependent with respect to different growth substrates. Our CRISPR screens establish parameters for genome-wide screens in hPSCs, which will facilitate the characterization of unappreciated genetic regulators of hPSC biology.
Asunto(s)
Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas/genética , Regulación de la Expresión Génica/genética , Genes Esenciales/genética , Células Madre Pluripotentes/metabolismo , Diferenciación Celular , HumanosRESUMEN
Genetic interactions identify combinations of genetic variants that impinge on phenotype. With whole-genome sequence information available for thousands of individuals within a species, a major outstanding issue concerns the interpretation of allelic combinations of genes underlying inherited traits. In this Review, we discuss how large-scale analyses in model systems have illuminated the general principles and phenotypic impact of genetic interactions. We focus on studies in budding yeast, including the mapping of a global genetic network. We emphasize how information gained from work in yeast translates to other systems, and how a global genetic network not only annotates gene function but also provides new insights into the genotype-to-phenotype relationship.
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Redes Reguladoras de Genes/genética , Redes Reguladoras de Genes/fisiología , Estudios de Asociación Genética/tendencias , Alelos , Animales , Frecuencia de los Genes/genética , Variación Genética/genética , Genotipo , Humanos , Modelos Genéticos , Fenotipo , Sitios de Carácter Cuantitativo/genética , Saccharomyces cerevisiae/genéticaRESUMEN
Histone lysine demethylases (KDMs) are of critical importance in the epigenetic regulation of gene expression, yet there are few selective, cell-permeable inhibitors or suitable tool compounds for these enzymes. We describe the discovery of a new class of inhibitor that is highly potent towards the histone lysine demethylases KDM2A/7A. A modular synthetic approach was used to explore the chemical space and accelerate the investigation of key structure-activity relationships, leading to the development of a small molecule with around 75-fold selectivity towards KDM2A/7A versus other KDMs, as well as cellular activity at low micromolar concentrations.
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Descubrimiento de Drogas , Inhibidores Enzimáticos/farmacología , Proteínas F-Box/antagonistas & inhibidores , Histona Demetilasas con Dominio de Jumonji/antagonistas & inhibidores , Relación Dosis-Respuesta a Droga , Inhibidores Enzimáticos/síntesis química , Inhibidores Enzimáticos/química , Proteínas F-Box/metabolismo , Células HeLa , Humanos , Histona Demetilasas con Dominio de Jumonji/metabolismo , Estructura Molecular , Relación Estructura-ActividadRESUMEN
Lung cancer is the leading cause of cancer deaths, and effective treatments are urgently needed. Loss-of-function mutations in the DNA damage response kinase ATM are common in lung adenocarcinoma but directly targeting these with drugs remains challenging. Here we report that ATM loss-of-function is synthetic lethal with drugs inhibiting the central growth factor kinases MEK1/2, including the FDA-approved drug trametinib. Lung cancer cells resistant to MEK inhibition become highly sensitive upon loss of ATM both in vitro and in vivo. Mechanistically, ATM mediates crosstalk between the prosurvival MEK/ERK and AKT/mTOR pathways. ATM loss also enhances the sensitivity of KRAS- or BRAF-mutant lung cancer cells to MEK inhibition. Thus, ATM mutational status in lung cancer is a mechanistic biomarker for MEK inhibitor response, which may improve patient stratification and extend the applicability of these drugs beyond RAS and BRAF mutant tumours.
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Proteínas de la Ataxia Telangiectasia Mutada/genética , Proliferación Celular/efectos de los fármacos , Neoplasias Pulmonares/prevención & control , Mutación , Inhibidores de Proteínas Quinasas/farmacología , Animales , Proteínas de la Ataxia Telangiectasia Mutada/metabolismo , Benzamidas/farmacología , Línea Celular Tumoral , Proliferación Celular/genética , Difenilamina/análogos & derivados , Difenilamina/farmacología , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Ratones Desnudos , Proteínas Proto-Oncogénicas B-raf/genética , Proteínas Proto-Oncogénicas B-raf/metabolismo , Piridonas/farmacología , Pirimidinonas/farmacología , Interferencia de ARN , Tiofenos/farmacología , Urea/análogos & derivados , Urea/farmacología , Ensayos Antitumor por Modelo de Xenoinjerto , Proteínas ras/genética , Proteínas ras/metabolismoRESUMEN
Patterns of somatic mutations in cancer genes provide information about their functional role in tumourigenesis, and thus indicate their potential for therapeutic exploitation. Yet, the classical distinction between oncogene and tumour suppressor may not always apply. For instance, TP53 has been simultaneously associated with tumour suppressing and promoting activities. Here, we uncover a similar phenomenon for GATA3, a frequently mutated, yet poorly understood, breast cancer gene. We identify two functional classes of frameshift mutations that are associated with distinct expression profiles in tumours, differential disease-free patient survival and gain- and loss-of-function activities in a cell line model. Furthermore, we find an estrogen receptor-independent synthetic lethal interaction between a GATA3 frameshift mutant with an extended C-terminus and the histone methyltransferases G9A and GLP, indicating perturbed epigenetic regulation. Our findings reveal important insights into mutant GATA3 function and breast cancer, provide the first potential therapeutic strategy and suggest that dual tumour suppressive and oncogenic activities are more widespread than previously appreciated.
Asunto(s)
Neoplasias de la Mama/genética , Epigénesis Genética , Factor de Transcripción GATA3/genética , Antígenos de Histocompatibilidad/genética , N-Metiltransferasa de Histona-Lisina/genética , Neoplasias de la Mama/patología , Supervivencia sin Enfermedad , Resistencia a Antineoplásicos/genética , Estrógenos/genética , Estrógenos/metabolismo , Femenino , Mutación del Sistema de Lectura , Regulación Neoplásica de la Expresión Génica , Humanos , Células MCF-7 , Bibliotecas de Moléculas Pequeñas/química , Bibliotecas de Moléculas Pequeñas/uso terapéuticoRESUMEN
Some mutations in cancer cells can be exploited for therapeutic intervention. However, for many cancer subtypes, including triple-negative breast cancer (TNBC), no frequently recurring aberrations could be identified to make such an approach clinically feasible. Characterized by a highly heterogeneous mutational landscape with few common features, many TNBCs cluster together based on their 'basal-like' transcriptional profiles. We therefore hypothesized that targeting TNBC cells on a systems level by exploiting the transcriptional cell state might be a viable strategy to find novel therapies for this highly aggressive disease. We performed a large-scale chemical genetic screen and identified a group of compounds related to the drug PKC412 (midostaurin). PKC412 induced apoptosis in a subset of TNBC cells enriched for the basal-like subtype and inhibited tumor growth in vivo. We employed a multi-omics approach and computational modeling to address the mechanism of action and identified spleen tyrosine kinase (SYK) as a novel and unexpected target in TNBC. Quantitative phosphoproteomics revealed that SYK inhibition abrogates signaling to STAT3, explaining the selectivity for basal-like breast cancer cells. This non-oncogene addiction suggests that chemical SYK inhibition may be beneficial for a specific subset of TNBC patients and demonstrates that targeting cell states could be a viable strategy to discover novel treatment strategies.
Asunto(s)
Antineoplásicos/farmacología , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Terapia Molecular Dirigida , Proteínas Tirosina Quinasas/metabolismo , Factor de Transcripción STAT3/metabolismo , Estaurosporina/análogos & derivados , Neoplasias de la Mama Triple Negativas/metabolismo , Animales , Apoptosis/efectos de los fármacos , Diferenciación Celular/efectos de los fármacos , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Sistemas de Liberación de Medicamentos , Femenino , Perfilación de la Expresión Génica , Humanos , Ratones , Simulación del Acoplamiento Molecular , Dominios y Motivos de Interacción de Proteínas , Proteómica/métodos , Análisis de Secuencia de ARN , Transducción de Señal , Estaurosporina/farmacología , Quinasa Syk , Factor de Crecimiento Transformador beta/genética , Factor de Crecimiento Transformador beta/metabolismo , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Epigenetic deregulation is a hallmark of cancer, and there has been increasing interest in therapeutics that target chromatin-modifying enzymes and other epigenetic regulators. The rationale for applying epigenetic drugs to treat cancer is twofold. First, epigenetic changes are reversible, and drugs could therefore be used to restore the normal (healthy) epigenetic landscape. However, it is unclear whether drugs can faithfully restore the precancerous epigenetic state. Second, chromatin regulators are often mutated in cancer, making them attractive drug targets. However, in most instances it is unknown whether cancer cells are addicted to these mutated chromatin proteins, or whether their mutation merely results in epigenetic instability conducive to the selection of secondary aberrations. An alternative incentive for targeting chromatin regulators is the exploitation of cancer-specific vulnerabilities, including synthetic lethality, caused by epigenetic deregulation. We review evidence for the hypothesis that mechanisms other than oncogene addiction are a basis for the application of epigenetic drugs, and propose future research directions.
Asunto(s)
Terapia Genética/métodos , Neoplasias/genética , Neoplasias/terapia , Epigenómica , HumanosRESUMEN
The process of tRNA splicing entails removal of an intron by TSEN (tRNA-splicing endonuclease) and ligation of the resulting exon halves to generate functional tRNAs. In mammalian cells, the RNA kinase CLP1 (cleavage and polyadenylation factor I subunit) associates with TSEN and phosphorylates the 3' exon at the 5' end in vitro, suggesting a role for CLP1 in tRNA splicing. Interestingly, recent data suggest that the ATP-binding and/or hydrolysis capacity of CLP1 is required to enhance pre-tRNA cleavage. In vivo, the lack of CLP1 kinase activity leads to progressive motor neuron loss and accumulation of novel 5' leader-5' exon tRNA fragments. We have extended the investigation of the biochemical requirements in pre-tRNA splicing and found that ß-γ-hydrolysable ATP is crucial for the productive generation of exon halves. In addition, we provide evidence that phosphorylation of the TSEN complex components supports efficient pre-tRNA cleavage. Taken together, our data improve the mechanistic understanding of mammalian pre-tRNA processing and its regulation.
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Adenosina Trifosfato/metabolismo , Endorribonucleasas/metabolismo , Intrones , Precursores del ARN/genética , Empalme del ARN , ARN de Transferencia/genética , Animales , Humanos , Hidrólisis , Ratones , FosforilaciónRESUMEN
CLP1 was the first mammalian RNA kinase to be identified. However, determining its in vivo function has been elusive. Here we generated kinase-dead Clp1 (Clp1(K/K)) mice that show a progressive loss of spinal motor neurons associated with axonal degeneration in the peripheral nerves and denervation of neuromuscular junctions, resulting in impaired motor function, muscle weakness, paralysis and fatal respiratory failure. Transgenic rescue experiments show that CLP1 functions in motor neurons. Mechanistically, loss of CLP1 activity results in accumulation of a novel set of small RNA fragments, derived from aberrant processing of tyrosine pre-transfer RNA. These tRNA fragments sensitize cells to oxidative-stress-induced p53 (also known as TRP53) activation and p53-dependent cell death. Genetic inactivation of p53 rescues Clp1(K/K) mice from the motor neuron loss, muscle denervation and respiratory failure. Our experiments uncover a mechanistic link between tRNA processing, formation of a new RNA species and progressive loss of lower motor neurons regulated by p53.
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Neuronas Motoras/metabolismo , Neuronas Motoras/patología , ARN de Transferencia de Tirosina/metabolismo , Factores de Transcripción/metabolismo , Esclerosis Amiotrófica Lateral , Animales , Animales Recién Nacidos , Axones/metabolismo , Axones/patología , Muerte Celular , Diafragma/inervación , Pérdida del Embrión , Embrión de Mamíferos/metabolismo , Embrión de Mamíferos/patología , Exones/genética , Femenino , Fibroblastos , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Atrofia Muscular Espinal , Enfermedades Neuromusculares/metabolismo , Enfermedades Neuromusculares/patología , Estrés Oxidativo , Procesamiento Postranscripcional del ARN , ARN de Transferencia de Tirosina/genética , Proteínas de Unión al ARN , Respiración , Nervios Espinales/citología , Factores de Transcripción/deficiencia , Proteína p53 Supresora de Tumor/metabolismo , Tirosina/genética , Tirosina/metabolismoRESUMEN
Although visual feedback (i.e. seeing our hand while we move it) improves the accuracy and efficiency of grasping movements, these positive effects of visual feedback are not consistently found for catching. It was the aim of our study to compare the efficiency of the use of visual feedback in grasping and catching and to explore possible reasons why visual feedback effects have been found less consistently in catching than in grasping. The first reason might be technical. Less sensitive measurement methods have been used in catching; this might explain why some catching studies did not find visual feedback effects. This problem was avoided in our study by using the same methods for both the catching and the grasping task. The effects of visual feedback were examined under standard conditions and under conditions where subjects wore prismatic glasses. Nevertheless, visual feedback effects were obtained for grasping but not for catching movements. This confirms that the difference in the use of visual feedback is real and not due to technical differences between grasping and catching studies. The second reason relates to the different temporal demands of grasping and catching. During a catching task, subjects have less time to respond, and therefore might not have sufficient time to use visual feedback. We tested this explanation with a task that required subjects to reach for a stationary object (i.e. grasping) as quickly as they had for the moving object in the catching task. However, even in this time-constrained grasping task, significant visual feedback effects were found, suggesting that time constraints do not explain the lack of visual feedback effects in catching. In our last explanation, we suggest that possibly the mode of motor control is different for catching and grasping, more particularly while grasping allows for on-line corrections, such corrections might not be possible for catching movements. We tested this explanation with a catching task that contained perturbation trials. During those perturbation trials, the target trajectory was shifted, after the subject had already started to move. We found that subjects responded to the target shifts with an appropriate shift of their catching response. This shows that on-line corrections are possible in the case of catching movements. We conclude that neither differences in the temporal demands nor in the capacity to make on-line modifications explain why visual feedback is used less effectively in the catching than in the grasping task.