RESUMEN
CMTR2 is an mRNA cap methyltransferase with poorly understood physiological functions. It catalyzes 2'-O-ribose methylation of the second transcribed nucleotide of mRNAs, potentially serving to mark RNAs as "self" to evade the cellular innate immune response. Here we analyze the consequences of Cmtr2 deficiency in mice. We discover that constitutive deletion of Cmtr2 results in mouse embryos that die during mid-gestation, exhibiting defects in embryo size, placental malformation and yolk sac vascularization. Endothelial cell deletion of Cmtr2 in mice results in vascular and hematopoietic defects, and perinatal lethality. Detailed characterization of the constitutive Cmtr2 KO phenotype shows an activation of the p53 pathway and decreased proliferation, but no evidence of interferon pathway activation. In summary, our study reveals the essential roles of Cmtr2 in mammalian cells beyond its immunoregulatory function.
RESUMEN
Receptor tyrosine kinase (RTK)-RAS signalling through the downstream mitogen-activated protein kinase (MAPK) cascade regulates cell proliferation and survival. The SHOC2-MRAS-PP1C holophosphatase complex functions as a key regulator of RTK-RAS signalling by removing an inhibitory phosphorylation event on the RAF family of proteins to potentiate MAPK signalling1. SHOC2 forms a ternary complex with MRAS and PP1C, and human germline gain-of-function mutations in this complex result in congenital RASopathy syndromes2-5. However, the structure and assembly of this complex are poorly understood. Here we use cryo-electron microscopy to resolve the structure of the SHOC2-MRAS-PP1C complex. We define the biophysical principles of holoenzyme interactions, elucidate the assembly order of the complex, and systematically interrogate the functional consequence of nearly all of the possible missense variants of SHOC2 through deep mutational scanning. We show that SHOC2 binds PP1C and MRAS through the concave surface of the leucine-rich repeat region and further engages PP1C through the N-terminal disordered region that contains a cryptic RVXF motif. Complex formation is initially mediated by interactions between SHOC2 and PP1C and is stabilized by the binding of GTP-loaded MRAS. These observations explain how mutant versions of SHOC2 in RASopathies and cancer stabilize the interactions of complex members to enhance holophosphatase activity. Together, this integrative structure-function model comprehensively defines key binding interactions within the SHOC2-MRAS-PP1C holophosphatase complex and will inform therapeutic development .
Asunto(s)
Microscopía por Crioelectrón , Péptidos y Proteínas de Señalización Intracelular , Complejos Multiproteicos , Proteína Fosfatasa 1 , Proteínas ras , Secuencias de Aminoácidos , Sitios de Unión , Guanosina Trifosfato/metabolismo , Humanos , Péptidos y Proteínas de Señalización Intracelular/química , Péptidos y Proteínas de Señalización Intracelular/genética , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Sistema de Señalización de MAP Quinasas , Complejos Multiproteicos/química , Complejos Multiproteicos/metabolismo , Complejos Multiproteicos/ultraestructura , Mutación Missense , Fosforilación , Unión Proteica , Proteína Fosfatasa 1/química , Proteína Fosfatasa 1/metabolismo , Proteína Fosfatasa 1/ultraestructura , Estabilidad Proteica , Quinasas raf , Proteínas ras/química , Proteínas ras/metabolismo , Proteínas ras/ultraestructuraRESUMEN
In a short hairpin RNA screen for genes that affect AKT phosphorylation, we identified the RAB35 small guanosine triphosphatase (GTPase)-a protein previously implicated in endomembrane trafficking-as a regulator of the phosphatidylinositol 3'-OH kinase (PI3K) pathway. Depletion of RAB35 suppresses AKT phosphorylation in response to growth factors, whereas expression of a dominant active GTPase-deficient mutant of RAB35 constitutively activates the PI3K/AKT pathway. RAB35 functions downstream of growth factor receptors and upstream of PDK1 and mTORC2 and copurifies with PI3K in immunoprecipitation assays. Two somatic RAB35 mutations found in human tumors generate alleles that constitutively activate PI3K/AKT signaling, suppress apoptosis, and transform cells in a PI3K-dependent manner. Furthermore, oncogenic RAB35 is sufficient to drive platelet-derived growth factor receptor α to LAMP2-positive endomembranes in the absence of ligand, suggesting that there may be latent oncogenic potential in dysregulated endomembrane trafficking.
Asunto(s)
Neoplasias/metabolismo , Proteínas Oncogénicas/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Proteínas de Unión al GTP rab/metabolismo , Alelos , Línea Celular Tumoral , Eliminación de Gen , Humanos , Inmunoprecipitación , Proteína 2 de la Membrana Asociada a los Lisosomas/metabolismo , Diana Mecanicista del Complejo 2 de la Rapamicina , Complejos Multiproteicos/metabolismo , Mutación , Neoplasias/genética , Neoplasias/patología , Proteínas Oncogénicas/genética , Fosforilación/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Transporte de Proteínas , Proteínas Proto-Oncogénicas c-akt/metabolismo , Piruvato Deshidrogenasa Quinasa Acetil-Transferidora , Interferencia de ARN , ARN Interferente Pequeño/genética , Receptor alfa de Factor de Crecimiento Derivado de Plaquetas/metabolismo , Serina-Treonina Quinasas TOR/metabolismo , Proteínas de Unión al GTP rab/genéticaRESUMEN
The evolutionarily conserved target of rapamycin complex 1 (TORC1) controls cell growth in response to nutrient availability and growth factors. TORC1 signaling is hyperactive in cancer, and regulators of TORC1 signaling may represent therapeutic targets for human diseases. To identify novel regulators of TORC1 signaling, we performed a genome-scale RNA interference screen on microarrays of Drosophila melanogaster cells expressing human RPS6, a TORC1 effector whose phosphorylated form we detected by immunofluorescence. Our screen revealed that the TORC1-S6K-RPS6 signaling axis is regulated by many subcellular components, including the Class I vesicle coat (COPI), the spliceosome, the proteasome, the nuclear pore, and the translation initiation machinery. Using additional RNAi reagents, we confirmed 70 novel genes as significant on-target regulators of RPS6 phosphorylation, and we characterized them with extensive secondary assays probing various arms of the TORC1 pathways, identifying functional relationships among those genes. We conclude that cell-based microarrays are a useful platform for genome-scale and secondary screening in Drosophila, revealing regulators that may represent drug targets for cancers and other diseases of deregulated TORC1 signaling.
Asunto(s)
Proteínas Recombinantes/metabolismo , Proteína S6 Ribosómica/metabolismo , Factores de Transcripción/metabolismo , Animales , Western Blotting , Células Cultivadas , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Técnica del Anticuerpo Fluorescente , Redes Reguladoras de Genes , Genoma , Genómica , Humanos , Análisis por Micromatrices , Terapia Molecular Dirigida , Fosforilación , Interferencia de ARN , Proteínas Recombinantes/genética , Proteína S6 Ribosómica/genética , Transducción de Señal/genética , Factores de Transcripción/genéticaRESUMEN
BACKGROUND: Image-based screens can produce hundreds of measured features for each of hundreds of millions of individual cells in a single experiment. RESULTS: Here, we describe CellProfiler Analyst, open-source software for the interactive exploration and analysis of multidimensional data, particularly data from high-throughput, image-based experiments. CONCLUSION: The system enables interactive data exploration for image-based screens and automated scoring of complex phenotypes that require combinations of multiple measured features per cell.
Asunto(s)
Células/ultraestructura , Biología Computacional/métodos , Procesamiento de Imagen Asistido por Computador/métodos , Fenotipo , Programas Informáticos , Inteligencia ArtificialRESUMEN
The recent development of cell microarrays offers the potential to accelerate high-throughput functional genetic studies. The widespread use of RNA interference (RNAi) has prompted several groups to fabricate RNAi cell microarrays that make possible discrete, in-parallel transfection with thousands of RNAi reagents on a microarray slide. Though still a budding technology, RNAi cell microarrays promise to increase the efficiency, economy and ease of genome-wide RNAi screens in metazoan cells.
Asunto(s)
Análisis de Secuencia por Matrices de Oligonucleótidos/métodos , Interferencia de ARN , Animales , Células Cultivadas , Silenciador del Gen , Genómica , Humanos , Procesamiento de Imagen Asistido por Computador , Análisis de Secuencia por Matrices de Oligonucleótidos/tendenciasRESUMEN
RNA interference (RNAi)-mediated loss-of-function screening in Drosophila melanogaster tissue culture cells is a powerful method for identifying the genes underlying cell biological functions and for annotating the fly genome. Here we describe the development of living-cell microarrays for screening large collections of RNAi-inducing double-stranded RNAs (dsRNAs) in Drosophila cells. The features of the microarrays consist of clusters of cells 200 mum in diameter, each with an RNAi-mediated depletion of a specific gene product. Because of the small size of the features, thousands of distinct dsRNAs can be screened on a single chip. The microarrays are suitable for quantitative and high-content cellular phenotyping and, in combination screens, for the identification of genetic suppressors, enhancers and synthetic lethal interactions. We used a prototype cell microarray with 384 different dsRNAs to identify previously unknown genes that affect cell proliferation and morphology, and, in a combination screen, that regulate dAkt/dPKB phosphorylation in the absence of dPTEN expression.