RESUMEN
Cells have evolved complex mechanisms to maintain protein homeostasis, such as the UPRER, which are strongly associated with several diseases and the aging process. We performed a whole-genome CRISPR-based knockout (KO) screen to identify genes important for cells to survive ER-based protein misfolding stress. We identified the cell-surface hyaluronidase (HAase), Transmembrane Protein 2 (TMEM2), as a potent modulator of ER stress resistance. The breakdown of the glycosaminoglycan, hyaluronan (HA), by TMEM2 within the extracellular matrix (ECM) altered ER stress resistance independent of canonical UPRER pathways but dependent upon the cell-surface receptor, CD44, a putative HA receptor, and the MAPK cell-signaling components, ERK and p38. Last, and most surprisingly, ectopic expression of human TMEM2 in C. elegans protected animals from ER stress and increased both longevity and pathogen resistance independent of canonical UPRER activation but dependent on the ERK ortholog mpk-1 and the p38 ortholog pmk-1.
Asunto(s)
Caenorhabditis elegans/fisiología , Retículo Endoplásmico/metabolismo , Hialuronoglucosaminidasa/metabolismo , Longevidad/fisiología , Proteínas de la Membrana/metabolismo , Respuesta de Proteína Desplegada , Animales , Caenorhabditis elegans/inmunología , Línea Celular , Proliferación Celular , Resistencia a la Enfermedad , Estrés del Retículo Endoplásmico , Fibroblastos/metabolismo , Humanos , Inmunidad Innata , Modelos Biológicos , Peso Molecular , Transducción de SeñalRESUMEN
Genetic screens are powerful tools for identifying genes responsible for diverse phenotypes. Here we describe a genome-wide CRISPR/Cas9-mediated loss-of-function screen in tumor growth and metastasis. We mutagenized a non-metastatic mouse cancer cell line using a genome-scale library with 67,405 single-guide RNAs (sgRNAs). The mutant cell pool rapidly generates metastases when transplanted into immunocompromised mice. Enriched sgRNAs in lung metastases and late-stage primary tumors were found to target a small set of genes, suggesting that specific loss-of-function mutations drive tumor growth and metastasis. Individual sgRNAs and a small pool of 624 sgRNAs targeting the top-scoring genes from the primary screen dramatically accelerate metastasis. In all of these experiments, the effect of mutations on primary tumor growth positively correlates with the development of metastases. Our study demonstrates Cas9-based screening as a robust method to systematically assay gene phenotypes in cancer evolution in vivo.
Asunto(s)
Sistemas CRISPR-Cas , Carcinoma de Pulmón de Células no Pequeñas/genética , Técnicas de Inactivación de Genes , Neoplasias Pulmonares/genética , Metástasis de la Neoplasia/genética , Animales , Carcinoma de Pulmón de Células no Pequeñas/patología , Estudio de Asociación del Genoma Completo , Humanos , Neoplasias Pulmonares/patología , Ratones , ARN Guía de KinetoplastidaRESUMEN
Finding the components of cellular circuits and determining their functions systematically remains a major challenge in mammalian cells. Here, we introduced genome-wide pooled CRISPR-Cas9 libraries into dendritic cells (DCs) to identify genes that control the induction of tumor necrosis factor (Tnf) by bacterial lipopolysaccharide (LPS), a key process in the host response to pathogens, mediated by the Tlr4 pathway. We found many of the known regulators of Tlr4 signaling, as well as dozens of previously unknown candidates that we validated. By measuring protein markers and mRNA profiles in DCs that are deficient in known or candidate genes, we classified the genes into three functional modules with distinct effects on the canonical responses to LPS and highlighted functions for the PAF complex and oligosaccharyltransferase (OST) complex. Our findings uncover new facets of innate immune circuits in primary cells and provide a genetic approach for dissection of mammalian cell circuits.
Asunto(s)
Sistemas CRISPR-Cas , Técnicas Genéticas , Inmunidad Innata , Animales , Células de la Médula Ósea/inmunología , Diferenciación Celular , Supervivencia Celular , Células Dendríticas/citología , Células Dendríticas/inmunología , Técnicas de Inactivación de Genes , Redes Reguladoras de Genes , Hexosiltransferasas/metabolismo , Proteínas de la Membrana/metabolismo , Ratones , Ratones Transgénicos , Receptor Toll-Like 4/inmunología , Factor de Necrosis Tumoral alfa/inmunologíaRESUMEN
Mutational patterns caused by APOBEC3 cytidine deaminase activity are evident throughout human cancer genomes. In particular, the APOBEC3A family member is a potent genotoxin that causes substantial DNA damage in experimental systems and human tumors. However, the mechanisms that ensure genome stability in cells with active APOBEC3A are unknown. Through an unbiased genome-wide screen, we define the Structural Maintenance of Chromosomes 5/6 (SMC5/6) complex as essential for cell viability when APOBEC3A is active. We observe an absence of APOBEC3A mutagenesis in human tumors with SMC5/6 dysfunction, consistent with synthetic lethality. Cancer cells depleted of SMC5/6 incur substantial genome damage from APOBEC3A activity during DNA replication. Further, APOBEC3A activity results in replication tract lengthening which is dependent on PrimPol, consistent with re-initiation of DNA synthesis downstream of APOBEC3A-induced lesions. Loss of SMC5/6 abrogates elongated replication tracts and increases DNA breaks upon APOBEC3A activity. Our findings indicate that replication fork lengthening reflects a DNA damage response to APOBEC3A activity that promotes genome stability in an SMC5/6-dependent manner. Therefore, SMC5/6 presents a potential therapeutic vulnerability in tumors with active APOBEC3A.
Asunto(s)
Proteínas de Ciclo Celular , Proteínas Cromosómicas no Histona , Citidina Desaminasa , Daño del ADN , Replicación del ADN , Humanos , Citidina Desaminasa/metabolismo , Citidina Desaminasa/genética , Proteínas de Ciclo Celular/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas Cromosómicas no Histona/metabolismo , Proteínas Cromosómicas no Histona/genética , Inestabilidad Genómica , Línea Celular Tumoral , ProteínasRESUMEN
The presence of large protein inclusions is a hallmark of neurodegeneration, and yet the precise molecular factors that contribute to their formation remain poorly understood. Screens using aggregation-prone proteins have commonly relied on downstream toxicity as a readout rather than the direct formation of aggregates. Here, we combined a genome-wide CRISPR knockout screen with Pulse Shape Analysis, a FACS-based method for inclusion detection, to identify direct modifiers of TDP-43 aggregation in human cells. Our screen revealed both canonical and novel proteostasis genes, and unearthed SRRD, a poorly characterized protein, as a top regulator of protein inclusion formation. APEX biotin labeling reveals that SRRD resides in proximity to proteins that are involved in the formation and breakage of disulfide bonds and to intermediate filaments, suggesting a role in regulation of the spatial dynamics of the intermediate filament network. Indeed, loss of SRRD results in aberrant intermediate filament fibrils and the impaired formation of aggresomes, including blunted vimentin cage structure, during proteotoxic stress. Interestingly, SRRD also localizes to aggresomes and unfolded proteins, and rescues proteotoxicity in yeast whereby its N-terminal low complexity domain is sufficient to induce this affect. Altogether this suggests an unanticipated and broad role for SRRD in cytoskeletal organization and cellular proteostasis.
Asunto(s)
Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas , Filamentos Intermedios , Humanos , Filamentos Intermedios/genética , Filamentos Intermedios/metabolismo , Citoesqueleto/genética , Cuerpos de Inclusión/genética , Cuerpos de Inclusión/metabolismoRESUMEN
Protein quality control pathways have evolved to ensure the fidelity of protein synthesis and efficiently clear potentially toxic protein species. Defects in ribosome-associated quality control and its associated factors have been implicated in the accumulation of aberrant proteins and neurodegeneration. C9orf72 repeat-associated non-AUG translation has been suggested to involve inefficient translation elongation, lead to ribosomal pausing and activation of ribosome-associated quality control pathways. However, the role of the ribosome-associated quality control complex in the processing of proteins generated through this non-canonical translation is not well understood. Here we use reporter constructs containing the C9orf72-associated hexanucleotide repeat, ribosome-associated quality control complex deficient cell models and stain for ribosome-associated quality control markers in C9orf72-expansion carrier human tissue to understand its role in dipeptide-repeat protein pathology. Our studies show that canonical ribosome-associated quality control substrates products are efficiently cleared by the ribosome-associated quality control complex in mammalian cells. Furthermore, using stalling reporter constructs, we show that repeats associated with the C9orf72-expansion induce ribosomal stalling when arginine (R)-rich dipeptide-repeat proteins are synthesized in a length-dependent manner. However, despite triggering this pathway, these arginine-rich dipeptide-repeat proteins are not efficiently processed by the core components of the ribosome-associated quality control complex (listerin, nuclear-export mediator factor and valosin containing protein) partly due to lack of lysine residues, which precludes ubiquitination. Deficient processing by this complex may be implicated in C9orf72-expansion associated disease as dipeptide-repeat protein inclusions were observed to be predominantly devoid of ubiquitin and co-localize with nuclear-export mediator factor in mutation carriers' frontal cortex and cerebellum tissue. These findings suggest that impaired processing of these arginine-rich dipeptide-repeat proteins derived from repeat-associated non-AUG translation by the ribosome-associated quality control complex may contribute to protein homeostasis dysregulation observed in C9orf72-expansion amyotrophic lateral sclerosis and frontotemporal degeneration neuropathogenesis.
Asunto(s)
Esclerosis Amiotrófica Lateral , Demencia Frontotemporal , Enfermedades Neurodegenerativas , Animales , Humanos , Dipéptidos/genética , Proteína C9orf72/genética , Proteína C9orf72/metabolismo , Esclerosis Amiotrófica Lateral/metabolismo , Enfermedades Neurodegenerativas/genética , Ribosomas , Expansión de las Repeticiones de ADN/genética , Demencia Frontotemporal/genética , Mamíferos/genética , Mamíferos/metabolismoRESUMEN
Recurrent chromosomal translocations producing a chimaeric MLL oncogene give rise to a highly aggressive acute leukaemia associated with poor clinical outcome. The preferential involvement of chromatin-associated factors as MLL fusion partners belies a dependency on transcription control. Despite recent progress made in targeting chromatin regulators in cancer, available therapies for this well-characterized disease remain inadequate, prompting the need to identify new targets for therapeutic intervention. Here, using unbiased CRISPR-Cas9 technology to perform a genome-scale loss-of-function screen in an MLL-AF4-positive acute leukaemia cell line, we identify ENL as an unrecognized gene that is specifically required for proliferation in vitro and in vivo. To explain the mechanistic role of ENL in leukaemia pathogenesis and dynamic transcription control, a chemical genetic strategy was developed to achieve targeted protein degradation. Acute loss of ENL suppressed the initiation and elongation of RNA polymerase II at active genes genome-wide, with pronounced effects at genes featuring a disproportionate ENL load. Notably, an intact YEATS chromatin-reader domain was essential for ENL-dependent leukaemic growth. Overall, these findings identify a dependency factor in acute leukaemia and suggest a mechanistic rationale for disrupting the YEATS domain in disease.
Asunto(s)
Regulación Neoplásica de la Expresión Génica , Leucemia/genética , Leucemia/metabolismo , Dominios Proteicos , Transcripción Genética , Factores de Elongación Transcripcional/química , Factores de Elongación Transcripcional/metabolismo , Animales , Sistemas CRISPR-Cas , Línea Celular Tumoral , Proliferación Celular , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Epigénesis Genética , Edición Génica , Genoma/genética , N-Metiltransferasa de Histona-Lisina/metabolismo , Humanos , Leucemia/patología , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/metabolismo , Leucemia Mieloide Aguda/patología , Ratones , Proteína de la Leucemia Mieloide-Linfoide/metabolismo , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/metabolismo , Leucemia-Linfoma Linfoblástico de Células Precursoras/patología , Proteolisis , ARN Polimerasa II/metabolismo , Elongación de la Transcripción Genética , Factores de Transcripción/química , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Factores de Elongación Transcripcional/genéticaRESUMEN
Somatic gene mutations can alter the vulnerability of cancer cells to T-cell-based immunotherapies. Here we perturbed genes in human melanoma cells to mimic loss-of-function mutations involved in resistance to these therapies, by using a genome-scale CRISPR-Cas9 library that consisted of around 123,000 single-guide RNAs, and profiled genes whose loss in tumour cells impaired the effector function of CD8+ T cells. The genes that were most enriched in the screen have key roles in antigen presentation and interferon-γ signalling, and correlate with cytolytic activity in patient tumours from The Cancer Genome Atlas. Among the genes validated using different cancer cell lines and antigens, we identified multiple loss-of-function mutations in APLNR, encoding the apelin receptor, in patient tumours that were refractory to immunotherapy. We show that APLNR interacts with JAK1, modulating interferon-γ responses in tumours, and that its functional loss reduces the efficacy of adoptive cell transfer and checkpoint blockade immunotherapies in mouse models. Our results link the loss of essential genes for the effector function of CD8+ T cells with the resistance or non-responsiveness of cancer to immunotherapies.
Asunto(s)
Genes Esenciales/genética , Inmunoterapia , Neoplasias/genética , Neoplasias/terapia , Linfocitos T Citotóxicos/efectos de los fármacos , Linfocitos T Citotóxicos/inmunología , Traslado Adoptivo , Animales , Presentación de Antígeno/genética , Apelina/metabolismo , Receptores de Apelina/genética , Receptores de Apelina/metabolismo , Sistemas CRISPR-Cas/genética , Línea Celular Tumoral , Femenino , Genoma/genética , Antígenos de Histocompatibilidad Clase I/inmunología , Humanos , Interferón gamma/inmunología , Janus Quinasa 1/metabolismo , Bases del Conocimiento , Melanoma/genética , Melanoma/inmunología , Melanoma/metabolismo , Melanoma/terapia , Ratones , Mutación , Neoplasias/inmunología , Neoplasias/metabolismo , Reproducibilidad de los Resultados , Linfocitos T Citotóxicos/metabolismoRESUMEN
Genome editing tools have simplified the generation of knock-in gene fusions, yet the prevalent use of gene-specific homology-directed repair (HDR) templates still hinders scalability. Consequently, realization of large-scale gene tagging requires further development of approaches to generate knock-in protein fusions via generic donors that do not require locus-specific homology sequences. Here, we combine intron-based protein trapping with homology-independent repair-based integration of a generic donor and demonstrate precise, scalable, and efficient gene tagging. Because editing is performed in introns using a synthetic exon, this approach tolerates mutations in the unedited allele, indels at the integration site, and the addition of resistance genes that do not disrupt the target gene coding sequence, resulting in easy and flexible gene tagging.
Asunto(s)
Edición Génica/métodos , Genoma Humano , Intrones , Mutagénesis Insercional , Proteínas Recombinantes de Fusión/genética , Secuencia de Bases , Proteína 9 Asociada a CRISPR/genética , Proteína 9 Asociada a CRISPR/metabolismo , Calnexina/genética , Calnexina/metabolismo , Línea Celular Tumoral , Homólogo de la Proteína Chromobox 5 , Proteínas Cromosómicas no Histona/genética , Proteínas Cromosómicas no Histona/metabolismo , Exones , Fibroblastos/citología , Fibroblastos/metabolismo , Expresión Génica , Células HEK293 , Células HeLa , Humanos , Plásmidos/química , Plásmidos/metabolismo , ARN Guía de Kinetoplastida/genética , ARN Guía de Kinetoplastida/metabolismo , Proteínas Recombinantes de Fusión/biosíntesis , Vimentina/genética , Vimentina/metabolismoRESUMEN
Forward genetic screens are powerful tools for the discovery and functional annotation of genetic elements. Recently, the RNA-guided CRISPR (clustered regularly interspaced short palindromic repeat)-associated Cas9 nuclease has been combined with genome-scale guide RNA libraries for unbiased, phenotypic screening. In this Review, we describe recent advances using Cas9 for genome-scale screens, including knockout approaches that inactivate genomic loci and strategies that modulate transcriptional activity. We discuss practical aspects of screen design, provide comparisons with RNA interference (RNAi) screening, and outline future applications and challenges.
Asunto(s)
Sistemas CRISPR-Cas , Genómica/métodos , Animales , Técnicas de Inactivación de Genes , Biblioteca de Genes , Pruebas Genéticas/métodos , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Humanos , Ratones , Modelos Genéticos , Interferencia de ARNRESUMEN
The RNA-guided endonuclease Cas9 has emerged as a versatile genome-editing platform. However, the size of the commonly used Cas9 from Streptococcus pyogenes (SpCas9) limits its utility for basic research and therapeutic applications that use the highly versatile adeno-associated virus (AAV) delivery vehicle. Here, we characterize six smaller Cas9 orthologues and show that Cas9 from Staphylococcus aureus (SaCas9) can edit the genome with efficiencies similar to those of SpCas9, while being more than 1 kilobase shorter. We packaged SaCas9 and its single guide RNA expression cassette into a single AAV vector and targeted the cholesterol regulatory gene Pcsk9 in the mouse liver. Within one week of injection, we observed >40% gene modification, accompanied by significant reductions in serum Pcsk9 and total cholesterol levels. We further assess the genome-wide targeting specificity of SaCas9 and SpCas9 using BLESS, and demonstrate that SaCas9-mediated in vivo genome editing has the potential to be efficient and specific.
Asunto(s)
Proteínas Asociadas a CRISPR/metabolismo , Ingeniería Genética/métodos , Genoma/genética , Staphylococcus aureus/enzimología , Animales , Secuencia de Bases , Proteínas Asociadas a CRISPR/genética , Colesterol/sangre , Colesterol/metabolismo , Marcación de Gen , Hígado/metabolismo , Hígado/fisiología , Masculino , Ratones , Ratones Endogámicos C57BL , Proproteína Convertasa 9 , Proproteína Convertasas/biosíntesis , Proproteína Convertasas/sangre , Proproteína Convertasas/deficiencia , Proproteína Convertasas/genética , Serina Endopeptidasas/biosíntesis , Serina Endopeptidasas/sangre , Serina Endopeptidasas/deficiencia , Serina Endopeptidasas/genética , Staphylococcus aureus/genética , Especificidad por SustratoRESUMEN
Enhancers, critical determinants of cellular identity, are commonly recognized by correlative chromatin marks and gain-of-function potential, although only loss-of-function studies can demonstrate their requirement in the native genomic context. Previously, we identified an erythroid enhancer of human BCL11A, subject to common genetic variation associated with the fetal haemoglobin level, the mouse orthologue of which is necessary for erythroid BCL11A expression. Here we develop pooled clustered regularly interspaced palindromic repeat (CRISPR)-Cas9 guide RNA libraries to perform in situ saturating mutagenesis of the human and mouse enhancers. This approach reveals critical minimal features and discrete vulnerabilities of these enhancers. Despite conserved function of the composite enhancers, their architecture diverges. The crucial human sequences appear to be primate-specific. Through editing of primary human progenitors and mouse transgenesis, we validate the BCL11A erythroid enhancer as a target for fetal haemoglobin reinduction. The detailed enhancer map will inform therapeutic genome editing, and the screening approach described here is generally applicable to functional interrogation of non-coding genomic elements.
Asunto(s)
Proteínas Asociadas a CRISPR/metabolismo , Proteínas Portadoras/genética , Elementos de Facilitación Genéticos/genética , Ingeniería Genética , Mutagénesis/genética , Proteínas Nucleares/genética , Animales , Secuencia de Bases , Sistemas CRISPR-Cas/genética , Células Cultivadas , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas/genética , Proteínas de Unión al ADN , Eritroblastos/metabolismo , Hemoglobina Fetal/genética , Genoma/genética , Humanos , Ratones , Datos de Secuencia Molecular , Especificidad de Órganos , ARN Guía de Kinetoplastida/genética , Proteínas Represoras , Reproducibilidad de los Resultados , Especificidad de la EspecieRESUMEN
CRISPR saturation mutagenesis has the potential to dissect the functional landscape of noncoding regions, but is highly susceptible to false discovery and misinterpretation. As recently published, Canver et al. have now taken the first steps towards addressing these issues by increasing screening resolution and analyzing the effects of off targets on hit calling.
Asunto(s)
Sistemas CRISPR-Cas , Genoma Humano , HumanosRESUMEN
The 3'end genomic region encodes a wide range of regulatory process including mRNA stability, 3' end processing and translation. Here, we systematically investigate the sequence determinants of 3' end mediated expression control by measuring the effect of 13,000 designed 3' end sequence variants on constitutive expression levels in yeast. By including a high resolution scanning mutagenesis of more than 200 native 3' end sequences in this designed set, we found that most mutations had only a mild effect on expression, and that the vast majority (~90%) of strongly effecting mutations localized to a single positive TA-rich element, similar to a previously described 3' end processing efficiency element, and resulted in up to ten-fold decrease in expression. Measurements of 3' UTR lengths revealed that these mutations result in mRNAs with aberrantly long 3'UTRs, confirming the role for this element in 3' end processing. Interestingly, we found that other sequence elements that were previously described in the literature to be part of the polyadenylation signal had a minor effect on expression. We further characterize the sequence specificities of the TA-rich element using additional synthetic 3' end sequences and show that its activity is sensitive to single base pair mutations and strongly depends on the A/T content of the surrounding sequences. Finally, using a computational model, we show that the strength of this element in native 3' end sequences can explain some of their measured expression variability (R = 0.41). Together, our results emphasize the importance of efficient 3' end processing for endogenous protein levels and contribute to an improved understanding of the sequence elements involved in this process.
Asunto(s)
Regiones no Traducidas 3' , Regulación Fúngica de la Expresión Génica , Levaduras/genética , Genoma Fúngico , ARN Mensajero/genética , ARN Mensajero/metabolismo , Levaduras/metabolismoRESUMEN
Retrograde axonal injury signalling stimulates cell body responses in lesioned peripheral neurons. The involvement of importins in retrograde transport suggests that transcription factors (TFs) might be directly involved in axonal injury signalling. Here, we show that multiple TFs are found in axons and associate with dynein in axoplasm from injured nerve. Biochemical and functional validation for one TF family establishes that axonal STAT3 is locally translated and activated upon injury, and is transported retrogradely with dynein and importin α5 to modulate survival of peripheral sensory neurons after injury. Hence, retrograde transport of TFs from axonal lesion sites provides a direct link between axon and nucleus.
Asunto(s)
Axones/metabolismo , Ganglios Espinales/metabolismo , Traumatismos de los Nervios Periféricos/metabolismo , Células Receptoras Sensoriales/metabolismo , Factores de Transcripción/metabolismo , Animales , Transporte Axonal/fisiología , Núcleo Celular/metabolismo , Dineínas/metabolismo , Carioferinas/metabolismo , Masculino , Ratones , Transporte de Proteínas/fisiología , Ratas , Ratas Wistar , Factor de Transcripción STAT3/metabolismo , Transducción de Señal/fisiologíaRESUMEN
A full understanding of gene regulation requires an understanding of the contributions that the various regulatory regions have on gene expression. Although it is well established that sequences downstream of the main promoter can affect expression, our understanding of the scale of this effect and how it is encoded in the DNA is limited. Here, to measure the effect of native S. cerevisiae 3' end sequences on expression, we constructed a library of 85 fluorescent reporter strains that differ only in their 3' end region. Notably, despite being driven by the same strong promoter, our library spans a continuous twelve-fold range of expression values. These measurements correlate with endogenous mRNA levels, suggesting that the 3' end contributes to constitutive differences in mRNA levels. We used deep sequencing to map the 3'UTR ends of our strains and show that determination of polyadenylation sites is intrinsic to the local 3' end sequence. Polyadenylation mapping was followed by sequence analysis, we found that increased A/T content upstream of the main polyadenylation site correlates with higher expression, both in the library and genome-wide, suggesting that native genes differ by the encoded efficiency of 3' end processing. Finally, we use single cells fluorescence measurements, in different promoter activation levels, to show that 3' end sequences modulate protein expression dynamics differently than promoters, by predominantly affecting the size of protein production bursts as opposed to the frequency at which these bursts occur. Altogether, our results lead to a more complete understanding of gene regulation by demonstrating that 3' end regions have a unique and sequence dependent effect on gene expression.
Asunto(s)
Regiones no Traducidas 3' , Regulación Fúngica de la Expresión Génica , ARN Mensajero/genética , ARN Mensajero/metabolismo , Composición de Base , Biología Computacional , Genes Fúngicos , Genes Reporteros , Poli A/genética , Poli A/metabolismo , Regiones Promotoras Genéticas , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismoRESUMEN
Transcriptome dynamics is governed by two opposing processes, mRNA production and degradation. Recent studies found that changes in these processes are frequently coordinated and that the relationship between them shapes transcriptome kinetics. Specifically, when transcription changes are counter-acted with changes in mRNA stability, transient fast-relaxing transcriptome kinetics is observed. A possible molecular mechanism underlying such coordinated regulation might lay in two RNA polymerase (Pol II) subunits, Rpb4 and Rpb7, which are recruited to mRNAs during transcription and later affect their degradation in the cytoplasm. Here we used a yeast strain carrying a mutant Pol II which poorly recruits these subunits. We show that this mutant strain is impaired in its ability to modulate mRNA stability in response to stress. The normal negative coordinated regulation is lost in the mutant, resulting in abnormal transcriptome profiles both with respect to magnitude and kinetics of responses. These results reveal an important role for Pol II, in regulation of both mRNA synthesis and degradation, and also in coordinating between them. We propose a simple model for production-degradation coupling that accounts for our observations. The model shows how a simple manipulation of the rates of co-transcriptional mRNA imprinting by Pol II may govern genome-wide transcriptome kinetics in response to environmental changes.
Asunto(s)
ARN Polimerasa II/fisiología , ARN Mensajero/biosíntesis , Proteínas de Saccharomyces cerevisiae/fisiología , Saccharomyces cerevisiae/genética , Transcriptoma , ARN Polimerasas Dirigidas por ADN/genética , Genoma Fúngico , Peróxido de Hidrógeno/toxicidad , Cinética , ARN Polimerasa II/genética , Estabilidad del ARN/genética , ARN Mensajero/genética , Proteínas de Saccharomyces cerevisiae/genética , Estrés Fisiológico/genética , Transcripción GenéticaRESUMEN
The ability to study proteins in a cellular context is crucial to our understanding of biology. Here, we report a new technology for "intracellular protein editing", drawing from intein- mediated protein splicing, genetic code expansion, and endogenous protein tagging. This protein editing approach enables us to rapidly and site specifically install residues and chemical handles into a protein of interest. We demonstrate the power of this protein editing platform to edit cellular proteins, inserting epitope peptides, protein-specific sequences, and non-canonical amino acids (ncAAs). Importantly, we employ an endogenous tagging approach to apply our protein editing technology to endogenous proteins with minimal perturbation. We anticipate that the protein editing technology presented here will be applied to a diverse set of problems, enabling novel experiments in live mammalian cells and therefore provide unique biological insights.
RESUMEN
Alpha-synuclein (αSyn) protein levels correlate with the risk and severity of Parkinson's disease and related neurodegenerative diseases. Lowering αSyn is being actively investigated as a therapeutic modality. Here, we systematically map the regulatory network that controls endogenous αSyn using sequential CRISPR-knockout and -interference screens in an αSyn gene (SNCA)-tagged cell line and induced pluripotent stem cell-derived neurons (iNeurons). We uncover αSyn modifiers at multiple regulatory layers, with amino-terminal acetyltransferase B (NatB) enzymes being the most potent endogenous αSyn modifiers in both cell lines. Amino-terminal acetylation protects the cytosolic αSyn from rapid degradation by the proteasome in a Ube2w-dependent manner. Moreover, we show that pharmacological inhibition of methionyl-aminopeptidase 2, a regulator of NatB complex formation, attenuates endogenous αSyn in iNeurons carrying SNCA triplication. Together, our study reveals several gene networks that control endogenous αSyn, identifies mechanisms mediating the degradation of nonacetylated αSyn, and illustrates potential therapeutic pathways for decreasing αSyn levels in synucleinopathies.
Asunto(s)
Acetiltransferasa B N-Terminal , Enfermedad de Parkinson , alfa-Sinucleína , Humanos , alfa-Sinucleína/genética , alfa-Sinucleína/metabolismo , Línea Celular , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas , Neuronas/metabolismo , Enfermedad de Parkinson/genética , Enfermedad de Parkinson/metabolismo , Acetiltransferasa B N-Terminal/antagonistas & inhibidores , Acetiltransferasa B N-Terminal/metabolismo , Metionil Aminopeptidasas/antagonistas & inhibidores , Metionil Aminopeptidasas/metabolismoRESUMEN
Mutational patterns caused by APOBEC3 cytidine deaminase activity are evident throughout human cancer genomes. In particular, the APOBEC3A family member is a potent genotoxin that causes substantial DNA damage in experimental systems and human tumors. However, the mechanisms that ensure genome stability in cells with active APOBEC3A are unknown. Through an unbiased genome-wide screen, we define the Structural Maintenance of Chromosomes 5/6 (SMC5/6) complex as essential for cell viability when APOBEC3A is active. We observe an absence of APOBEC3A mutagenesis in human tumors with SMC5/6 dysfunction, consistent with synthetic lethality. Cancer cells depleted of SMC5/6 incur substantial genome damage from APOBEC3A activity during DNA replication. Further, APOBEC3A activity results in replication tract lengthening which is dependent on PrimPol, consistent with re-initiation of DNA synthesis downstream of APOBEC3A-induced lesions. Loss of SMC5/6 abrogates elongated replication tracts and increases DNA breaks upon APOBEC3A activity. Our findings indicate that replication fork lengthening reflects a DNA damage response to APOBEC3A activity that promotes genome stability in an SMC5/6-dependent manner. Therefore, SMC5/6 presents a potential therapeutic vulnerability in tumors with active APOBEC3A.