RESUMO
CRISPR-Cas technology has transformed functional genomics, yet understanding of how individual exons differentially shape cellular phenotypes remains limited. Here, we optimized and conducted massively parallel exon deletion and splice-site mutation screens in human cell lines to identify exons that regulate cellular fitness. Fitness-promoting exons are prevalent in essential and highly expressed genes and commonly overlap with protein domains and interaction interfaces. Conversely, fitness-suppressing exons are enriched in nonessential genes, exhibiting lower inclusion levels, and overlap with intrinsically disordered regions and disease-associated mutations. In-depth mechanistic investigation of the screen-hit TAF5 alternative exon-8 revealed that its inclusion is required for assembly of the TFIID general transcription initiation complex, thereby regulating global gene expression output. Collectively, our orthogonal exon perturbation screens established a comprehensive repository of phenotypically important exons and uncovered regulatory mechanisms governing cellular fitness and gene expression.
Assuntos
Éxons , Humanos , Éxons/genética , Sistemas CRISPR-Cas , Fator de Transcrição TFIID/genética , Fator de Transcrição TFIID/metabolismo , Aptidão Genética , Células HEK293 , Fatores Associados à Proteína de Ligação a TATA/genética , Fatores Associados à Proteína de Ligação a TATA/metabolismo , Sítios de Splice de RNA , Mutação , Regulação da Expressão Gênica , Processamento AlternativoRESUMO
Chromosomal rearrangements have a central role in the pathogenesis of human cancers and often result in the expression of therapeutically actionable gene fusions. A recently discovered example is a fusion between the genes echinoderm microtubule-associated protein like 4 (EML4) and anaplastic lymphoma kinase (ALK), generated by an inversion on the short arm of chromosome 2: inv(2)(p21p23). The EML4-ALK oncogene is detected in a subset of human non-small cell lung cancers (NSCLC) and is clinically relevant because it confers sensitivity to ALK inhibitors. Despite their importance, modelling such genetic events in mice has proven challenging and requires complex manipulation of the germ line. Here we describe an efficient method to induce specific chromosomal rearrangements in vivo using viral-mediated delivery of the CRISPR/Cas9 system to somatic cells of adult animals. We apply it to generate a mouse model of Eml4-Alk-driven lung cancer. The resulting tumours invariably harbour the Eml4-Alk inversion, express the Eml4-Alk fusion gene, display histopathological and molecular features typical of ALK(+) human NSCLCs, and respond to treatment with ALK inhibitors. The general strategy described here substantially expands our ability to model human cancers in mice and potentially in other organisms.
Assuntos
Proteínas Associadas a CRISPR/genética , Sistemas CRISPR-Cas , Engenharia Genética/métodos , Translocação Genética/genética , Quinase do Linfoma Anaplásico , Animais , Antineoplásicos/uso terapêutico , Células Cultivadas , Inversão Cromossômica/genética , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Crizotinibe , Modelos Animais de Doenças , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/enzimologia , Neoplasias Pulmonares/patologia , Camundongos , Células NIH 3T3 , Inibidores de Proteínas Quinases/uso terapêutico , Pirazóis/uso terapêutico , Piridinas/uso terapêutico , Receptores Proteína Tirosina Quinases/metabolismoRESUMO
MicroRNAs repress mRNA translation by guiding Argonaute proteins to partially complementary binding sites, primarily within the 3' untranslated region (UTR) of target mRNAs. In cell lines, Argonaute-bound microRNAs exist mainly in high molecular weight RNA-induced silencing complexes (HMW-RISC) associated with target mRNA. Here we demonstrate that most adult tissues contain reservoirs of microRNAs in low molecular weight RISC (LMW-RISC) not bound to mRNA, suggesting that these microRNAs are not actively engaged in target repression. Consistent with this observation, the majority of individual microRNAs in primary T cells were enriched in LMW-RISC. During T-cell activation, signal transduction through the phosphoinositide-3 kinase-RAC-alpha serine/threonine-protein kinase-mechanistic target of rapamycin pathway increased the assembly of microRNAs into HMW-RISC, enhanced expression of the glycine-tryptophan protein of 182 kDa, an essential component of HMW-RISC, and improved the ability of microRNAs to repress partially complementary reporters, even when expression of targeting microRNAs did not increase. Overall, data presented here demonstrate that microRNA-mediated target repression in nontransformed cells depends not only on abundance of specific microRNAs, but also on regulation of RISC assembly by intracellular signaling.
Assuntos
Proteínas Argonautas/metabolismo , MicroRNAs/metabolismo , RNA Mensageiro/metabolismo , Ativação Linfocitária , Peso Molecular , Linfócitos T/metabolismoRESUMO
MicroRNAs belonging to the miR-34 family have been proposed as critical modulators of the p53 pathway and potential tumor suppressors in human cancers. To formally test these hypotheses, we have generated mice carrying targeted deletion of all three members of this microRNA family. We show that complete inactivation of miR-34 function is compatible with normal development in mice. Surprisingly, p53 function appears to be intact in miR-34-deficient cells and tissues. Although loss of miR-34 expression leads to a slight increase in cellular proliferation in vitro, it does not impair p53-induced cell cycle arrest or apoptosis. Furthermore, in contrast to p53-deficient mice, miR-34-deficient animals do not display increased susceptibility to spontaneous, irradiation-induced, or c-Myc-initiated tumorigenesis. We also show that expression of members of the miR-34 family is particularly high in the testes, lungs, and brains of mice and that it is largely p53-independent in these tissues. These findings indicate that miR-34 plays a redundant function in the p53 pathway and suggest additional p53-independent functions for this family of miRNAs.
Assuntos
Transformação Celular Neoplásica/genética , Regulação Neoplásica da Expressão Gênica , MicroRNAs/genética , Proteína Supressora de Tumor p53 , Animais , Apoptose/genética , Pontos de Checagem do Ciclo Celular/genética , Proliferação de Células , Células Cultivadas , Fibroblastos/citologia , Fibroblastos/metabolismo , Regulação Neoplásica da Expressão Gênica/genética , Humanos , Camundongos , Camundongos Knockout , MicroRNAs/metabolismo , Proteína Supressora de Tumor p53/deficiência , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismoRESUMO
Vault RNAs (vRNAs) are evolutionarily conserved small non-coding RNAs transcribed by RNA polymerase lll. Initially described as components of the vault particle, they have since also been described as noncanonical miRNA precursors and as riboregulators of autophagy. As central molecules in these processes, vRNAs have been attributed numerous biological roles including regulation of cell proliferation and survival, response to viral infections, drug resistance, and animal development. Yet, their impact to mammalian physiology remains largely unexplored. To study vault RNAs in vivo, we generated a mouse line with a conditional Vaultrc5 loss of function allele. Because Vaultrc5 is the sole murine vRNA, this allele enables the characterization of the physiological requirements of this conserved class of small regulatory RNAs in mammals. Using this strain, we show that mice constitutively null for Vaultrc5 are viable and histologically normal but have a slight reduction in platelet counts pointing to a potential role for vRNAs in hematopoiesis. This work paves the way for further in vivo characterizations of this abundant but mysterious RNA molecule. Specifically, it enables the study of the biological consequences of constitutive or lineage-specific Vaultrc5 deletion and of the physiological requirements for an intact Vaultrc5 during normal hematopoiesis or in response to cellular stresses such as oncogene expression, viral infection, or drug treatment.
RESUMO
The T-box transcription factor BRACHYURY (T) is a key regulator of mesoderm formation during early development. Complete loss of T has been shown to lead to embryonic lethality around E10.0. Here we characterize an inducible miRNA-based in vivo knockdown mouse model of T, termed KD3-T, which exhibits a hypomorphic phenotype. KD3-T embryos display axial skeletal defects caused by apoptosis of paraxial mesoderm, which is accompanied by urorectal malformations resembling the murine uro-recto-caudal syndrome and human caudal regression syndrome phenotypes. We show that there is a reduction of T in the notochord of KD3-T embryos which results in impaired notochord differentiation and its subsequent loss, whereas levels of T in the tailbud are sufficient for axis extension and patterning. Furthermore, the notochord in KD3-T embryos adopts a neural character and loses its ability to act as a signaling center. Since KD3-T animals survive until birth, they are useful for examining later roles for T in the development of urorectal tissues.
Assuntos
Anormalidades do Sistema Digestório/genética , Proteínas Fetais/genética , Siringomielia/genética , Proteínas com Domínio T/genética , Anormalidades Múltiplas , Canal Anal/anormalidades , Canal Anal/metabolismo , Animais , Apoptose , Diferenciação Celular , Anormalidades do Sistema Digestório/metabolismo , Modelos Animais de Doenças , Embrião de Mamíferos/metabolismo , Feminino , Proteínas Fetais/metabolismo , Imunofluorescência , Regulação da Expressão Gênica no Desenvolvimento , Meningocele , Camundongos , Camundongos Endogâmicos C57BL , MicroRNAs/metabolismo , Fenótipo , Reto/anormalidades , Reto/metabolismo , Região Sacrococcígea/anormalidades , Sacro/anormalidades , Sacro/metabolismo , Siringomielia/metabolismo , Proteínas com Domínio T/metabolismoRESUMO
Argonaute 2 (AGO2) is a cytoplasmic component of the miRNA pathway, with essential roles in development and disease. Yet little is known about its regulation in vivo. Here we show that in quiescent mouse splenocytes, AGO2 localizes almost exclusively to the nucleus. AGO2 subcellular localization is modulated by the Pi3K-AKT-mTOR pathway, a well-established regulator of quiescence. Signaling through this pathway in proliferating cells promotes AGO2 cytoplasmic accumulation, at least in part by stimulating the expression of TNRC6, an essential AGO2 binding partner in the miRNA pathway. In quiescent cells in which mTOR signaling is low, AGO2 accumulates in the nucleus, where it binds to young mobile transposons co-transcriptionally to repress their expression via its catalytic domain. Our data point to an essential but previously unrecognized nuclear role for AGO2 during quiescence as part of a genome-defense system against young mobile elements and provide evidence of RNA interference in the soma of mammals.
Assuntos
Proteínas Argonautas , MicroRNAs , Fosfatidilinositol 3-Quinases , Animais , Camundongos , Proteínas Argonautas/genética , Proteínas Argonautas/metabolismo , Linhagem Celular , Mamíferos/genética , MicroRNAs/metabolismo , Fosfatidilinositol 3-Quinases/genética , Fosfatidilinositol 3-Quinases/metabolismo , Interferência de RNA , Serina-Treonina Quinases TOR/genética , Serina-Treonina Quinases TOR/metabolismoRESUMO
Functional analysis of multiple genes is key to understanding gene regulatory networks controlling embryonic development. We have developed an integrated vector system for inducible gene silencing by shRNAmir-mediated RNA interference in mouse embryos, as a fast method for dissecting mammalian gene function. For validation of the vector system, we generated mutant phenotypes for Brachyury, Foxa2 and Noto, transcription factors which play pivotal roles in embryonic development. Using a series of Brachyury shRNAmir vectors of various strengths we generated hypomorphic and loss of function phenotypes allowing the identification of Brachyury target genes involved in trunk development. We also demonstrate temporal control of gene silencing, thus bypassing early embryonic lethality. Importantly, off-target effects of shRNAmir expression were not detectable. Taken together, the system allows the dissection of gene function at unprecedented detail and speed, and provides tight control of the genetic background minimizing intrinsic variation.
Assuntos
Desenvolvimento Embrionário/genética , Interferência de RNA , Animais , Células Cultivadas , Embrião de Mamíferos/metabolismo , Perfilação da Expressão Gênica , Vetores Genéticos , Camundongos , Camundongos Endogâmicos C57BL , Mutação , Fenótipo , Fatores de Transcrição/genética , TransgenesRESUMO
Off-target effects are well established confounders of CRISPR negative selection screens that impair the identification of essential genomic loci. In particular, non-coding regulatory elements and repetitive regions are often difficult to target with specific gRNAs, effectively precluding the unbiased screening of a large portion of the genome. To address this, we developed CRISPR Specificity Correction (CSC), a computational method that corrects for the effect of off-targeting on gRNA depletion. We benchmark CSC with data from the Cancer Dependency Map and show that it significantly improves the overall sensitivity and specificity of viability screens while preserving known essentialities, particularly for genes targeted by highly promiscuous gRNAs. We believe this tool will further enable the functional annotation of the genome as it represents a robust alternative to the traditional filtering strategy of discarding unspecific guides from the analysis. CSC is an open-source software that can be seamlessly integrated into current CRISPR analysis pipelines.
Assuntos
RNA Guia de Cinetoplastídeos/metabolismo , Sistemas CRISPR-Cas/genética , Sistemas CRISPR-Cas/fisiologia , Biologia Computacional/métodos , Edição de Genes , Humanos , RNA Guia de Cinetoplastídeos/genética , SoftwareRESUMO
Argonaute (AGO) proteins play key roles in animal physiology by binding to small RNAs and regulating the expression of their targets. In mammals, they do so through two distinct pathways: the miRNA pathway represses genes through a multiprotein complex that promotes both decay and translational repression; the siRNA pathway represses transcripts through direct Ago2-mediated cleavage. Here, we review our current knowledge of mechanistic details and physiological requirements of both these pathways and briefly discuss their implications to human disease.
Assuntos
Proteínas Argonautas/genética , Regulação da Expressão Gênica , MicroRNAs/genética , Família Multigênica/genética , RNA Interferente Pequeno/genética , Sequência de Aminoácidos , Animais , Proteínas Argonautas/química , Proteínas Argonautas/metabolismo , Humanos , MicroRNAs/metabolismo , Modelos Moleculares , Conformação Proteica , RNA Interferente Pequeno/metabolismoRESUMO
We present GuideScan software for the design of CRISPR guide RNA libraries that can be used to edit coding and noncoding genomic regions. GuideScan produces high-density sets of guide RNAs (gRNAs) for single- and paired-gRNA genome-wide screens. We also show that the trie data structure of GuideScan enables the design of gRNAs that are more specific than those designed by existing tools.
Assuntos
Algoritmos , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/genética , Inativação Gênica , Aprendizado de Máquina , RNA Interferente Pequeno/genética , Software , Sistemas CRISPR-Cas/genética , Mapeamento Cromossômico/métodos , Análise de Sequência de RNA/métodosRESUMO
Despite their clear importance as a class of regulatory molecules, pinpointing the relevance of individual miRNAs has been challenging. Studies querying miRNA functions by overexpressing or silencing specific miRNAs have yielded data that are often at odds with those collected from loss-of-functions models. In addition, knockout studies suggest that many conserved miRNAs are dispensable for animal development or viability. In this review, we discuss these observations in the context of our current knowledge of miRNA biology and review the evidence implicating miRNA-mediated gene regulation in the mechanisms that ensure biological robustness.
Assuntos
MicroRNAs/fisiologia , Interferência de RNA , Animais , Perfilação da Expressão Gênica , Redes Reguladoras de Genes , HumanosRESUMO
The CRISPR-Cas9 system is a powerful tool to edit eukaryotic genomes that has recently been adapted for functional screens. Several of its applications--including the disruption of genes using Cas9-nickase and the generation of large deletions--require co-expression of two distinct guide RNAs (gRNAs). However, the lack of experimental approaches to generate pools of paired gRNA vectors prevents these applications from being scalable. Here we report a simple, inexpensive, one-step method that allows for the rapid and efficient cloning of gRNA pairs into expression vectors. We show that this method can be used to generate pooled libraries and is therefore suitable for in vivo and in vitro functional screens.
Assuntos
Proteínas Associadas a CRISPR/metabolismo , Sistemas CRISPR-Cas/fisiologia , Biblioteca Gênica , RNA Guia de Cinetoplastídeos/genética , Animais , Proteínas Associadas a CRISPR/genética , Sistemas CRISPR-Cas/genética , Clonagem Molecular , Regulação da Expressão Gênica , Engenharia Genética/métodos , Células HEK293 , Humanos , Camundongos , Células NIH 3T3 , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismoRESUMO
Polycistronic microRNA (miRNA) clusters are a common feature of vertebrate genomes. The coordinated expression of miRNAs belonging to different seed families from a single transcriptional unit suggests functional cooperation, but this hypothesis has not been experimentally tested. Here we report the characterization of an allelic series of genetically engineered mice harboring selective targeted deletions of individual components of the miR-17 â¼ 92 cluster. Our results demonstrate the coexistence of functional cooperation and specialization among members of this cluster, identify a previously undescribed function for the miR-17 seed family in controlling axial patterning in vertebrates and show that loss of miR-19 selectively impairs Myc-driven tumorigenesis in two models of human cancer. By integrating phenotypic analysis and gene expression profiling, we provide a genome-wide view of how the components of a polycistronic miRNA cluster affect gene expression in vivo. The reagents and data sets reported here will accelerate exploration of the complex biological functions of this important miRNA cluster.