RESUMO
Analyses of ancient DNA typically involve sequencing the surviving short oligonucleotides and aligning to genome assemblies from related, modern species. Here, we report that skin from a female woolly mammoth (Mammuthus primigenius) that died 52,000 years ago retained its ancient genome architecture. We use PaleoHi-C to map chromatin contacts and assemble its genome, yielding 28 chromosome-length scaffolds. Chromosome territories, compartments, loops, Barr bodies, and inactive X chromosome (Xi) superdomains persist. The active and inactive genome compartments in mammoth skin more closely resemble Asian elephant skin than other elephant tissues. Our analyses uncover new biology. Differences in compartmentalization reveal genes whose transcription was potentially altered in mammoths vs. elephants. Mammoth Xi has a tetradic architecture, not bipartite like human and mouse. We hypothesize that, shortly after this mammoth's death, the sample spontaneously freeze-dried in the Siberian cold, leading to a glass transition that preserved subfossils of ancient chromosomes at nanometer scale.
Assuntos
Genoma , Mamutes , Pele , Animais , Mamutes/genética , Genoma/genética , Feminino , Elefantes/genética , Cromatina/genética , Fósseis , DNA Antigo/análise , Camundongos , Humanos , Cromossomo X/genéticaRESUMO
Systematic evaluation of the impact of genetic variants is critical for the study and treatment of human physiology and disease. While specific mutations can be introduced by genome engineering, we still lack scalable approaches that are applicable to the important setting of primary cells, such as blood and immune cells. Here, we describe the development of massively parallel base-editing screens in human hematopoietic stem and progenitor cells. Such approaches enable functional screens for variant effects across any hematopoietic differentiation state. Moreover, they allow for rich phenotyping through single-cell RNA sequencing readouts and separately for characterization of editing outcomes through pooled single-cell genotyping. We efficiently design improved leukemia immunotherapy approaches, comprehensively identify non-coding variants modulating fetal hemoglobin expression, define mechanisms regulating hematopoietic differentiation, and probe the pathogenicity of uncharacterized disease-associated variants. These strategies will advance effective and high-throughput variant-to-function mapping in human hematopoiesis to identify the causes of diverse diseases.
Assuntos
Edição de Genes , Células-Tronco Hematopoéticas , Humanos , Diferenciação Celular , Sistemas CRISPR-Cas , Genoma , Hematopoese , Células-Tronco Hematopoéticas/metabolismo , Engenharia Genética , Análise de Célula ÚnicaRESUMO
Single-cell (sc)RNA-seq, together with RNA velocity and metabolic labeling, reveals cellular states and transitions at unprecedented resolution. Fully exploiting these data, however, requires kinetic models capable of unveiling governing regulatory functions. Here, we introduce an analytical framework dynamo (https://github.com/aristoteleo/dynamo-release), which infers absolute RNA velocity, reconstructs continuous vector fields that predict cell fates, employs differential geometry to extract underlying regulations, and ultimately predicts optimal reprogramming paths and perturbation outcomes. We highlight dynamo's power to overcome fundamental limitations of conventional splicing-based RNA velocity analyses to enable accurate velocity estimations on a metabolically labeled human hematopoiesis scRNA-seq dataset. Furthermore, differential geometry analyses reveal mechanisms driving early megakaryocyte appearance and elucidate asymmetrical regulation within the PU.1-GATA1 circuit. Leveraging the least-action-path method, dynamo accurately predicts drivers of numerous hematopoietic transitions. Finally, in silico perturbations predict cell-fate diversions induced by gene perturbations. Dynamo, thus, represents an important step in advancing quantitative and predictive theories of cell-state transitions.
Assuntos
Análise de Célula Única , Transcriptoma/genética , Algoritmos , Feminino , Regulação da Expressão Gênica , Células HL-60 , Hematopoese/genética , Células-Tronco Hematopoéticas/metabolismo , Humanos , Cinética , Modelos Biológicos , RNA Mensageiro/metabolismo , Coloração e RotulagemRESUMO
Severe obesity is a rapidly growing global health threat. Although often attributed to unhealthy lifestyle choices or environmental factors, obesity is known to be heritable and highly polygenic; the majority of inherited susceptibility is related to the cumulative effect of many common DNA variants. Here we derive and validate a new polygenic predictor comprised of 2.1 million common variants to quantify this susceptibility and test this predictor in more than 300,000 individuals ranging from middle age to birth. Among middle-aged adults, we observe a 13-kg gradient in weight and a 25-fold gradient in risk of severe obesity across polygenic score deciles. In a longitudinal birth cohort, we note minimal differences in birthweight across score deciles, but a significant gradient emerged in early childhood and reached 12 kg by 18 years of age. This new approach to quantify inherited susceptibility to obesity affords new opportunities for clinical prevention and mechanistic assessment.
Assuntos
Peso Corporal , Herança Multifatorial/genética , Obesidade/patologia , Adolescente , Índice de Massa Corporal , Criança , Bases de Dados Factuais , Feminino , Estudo de Associação Genômica Ampla , Humanos , Recém-Nascido , Estudos Longitudinais , Masculino , Pessoa de Meia-Idade , Obesidade/genética , Fatores de Risco , Índice de Gravidade de DoençaRESUMO
Understanding the molecular programs that guide differentiation during development is a major challenge. Here, we introduce Waddington-OT, an approach for studying developmental time courses to infer ancestor-descendant fates and model the regulatory programs that underlie them. We apply the method to reconstruct the landscape of reprogramming from 315,000 single-cell RNA sequencing (scRNA-seq) profiles, collected at half-day intervals across 18 days. The results reveal a wider range of developmental programs than previously characterized. Cells gradually adopt either a terminal stromal state or a mesenchymal-to-epithelial transition state. The latter gives rise to populations related to pluripotent, extra-embryonic, and neural cells, with each harboring multiple finer subpopulations. The analysis predicts transcription factors and paracrine signals that affect fates and experiments validate that the TF Obox6 and the cytokine GDF9 enhance reprogramming efficiency. Our approach sheds light on the process and outcome of reprogramming and provides a framework applicable to diverse temporal processes in biology.
Assuntos
Reprogramação Celular/genética , Perfilação da Expressão Gênica/métodos , Análise de Célula Única/métodos , Animais , Diferenciação Celular/genética , Células Cultivadas , Células-Tronco Embrionárias/metabolismo , Fibroblastos/metabolismo , Expressão Gênica , Regulação da Expressão Gênica no Desenvolvimento/genética , Células-Tronco Pluripotentes Induzidas/metabolismo , Camundongos , Análise de Sequência de RNA/métodos , Fatores de Transcrição/metabolismoRESUMO
Intracellular accumulation of misfolded proteins causes toxic proteinopathies, diseases without targeted therapies. Mucin 1 kidney disease (MKD) results from a frameshift mutation in the MUC1 gene (MUC1-fs). Here, we show that MKD is a toxic proteinopathy. Intracellular MUC1-fs accumulation activated the ATF6 unfolded protein response (UPR) branch. We identified BRD4780, a small molecule that clears MUC1-fs from patient cells, from kidneys of knockin mice and from patient kidney organoids. MUC1-fs is trapped in TMED9 cargo receptor-containing vesicles of the early secretory pathway. BRD4780 binds TMED9, releases MUC1-fs, and re-routes it for lysosomal degradation, an effect phenocopied by TMED9 deletion. Our findings reveal BRD4780 as a promising lead for the treatment of MKD and other toxic proteinopathies. Generally, we elucidate a novel mechanism for the entrapment of misfolded proteins by cargo receptors and a strategy for their release and anterograde trafficking to the lysosome.
Assuntos
Benzamidas/metabolismo , Compostos Bicíclicos com Pontes/farmacologia , Heptanos/farmacologia , Lisossomos/efeitos dos fármacos , Proteínas de Transporte Vesicular/metabolismo , Fator 6 Ativador da Transcrição/metabolismo , Animais , Benzamidas/química , Benzamidas/farmacologia , Compostos Bicíclicos com Pontes/uso terapêutico , Células Epiteliais/citologia , Células Epiteliais/metabolismo , Feminino , Mutação da Fase de Leitura , Heptanos/uso terapêutico , Humanos , Receptores de Imidazolinas/antagonistas & inibidores , Receptores de Imidazolinas/genética , Receptores de Imidazolinas/metabolismo , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Pluripotentes Induzidas/metabolismo , Rim/citologia , Rim/metabolismo , Rim/patologia , Nefropatias/metabolismo , Nefropatias/patologia , Lisossomos/metabolismo , Masculino , Camundongos , Camundongos Transgênicos , Mucina-1/química , Mucina-1/genética , Mucina-1/metabolismo , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Resposta a Proteínas não Dobradas/efeitos dos fármacos , Proteínas de Transporte Vesicular/químicaRESUMO
Blood cell formation is classically thought to occur through a hierarchical differentiation process, although recent studies have shown that lineage commitment may occur earlier in hematopoietic stem and progenitor cells (HSPCs). The relevance to human blood diseases and the underlying regulation of these refined models remain poorly understood. By studying a genetic blood disorder, Diamond-Blackfan anemia (DBA), where the majority of mutations affect ribosomal proteins and the erythroid lineage is selectively perturbed, we are able to gain mechanistic insight into how lineage commitment is programmed normally and disrupted in disease. We show that in DBA, the pool of available ribosomes is limited, while ribosome composition remains constant. Surprisingly, this global reduction in ribosome levels more profoundly alters translation of a select subset of transcripts. We show how the reduced translation of select transcripts in HSPCs can impair erythroid lineage commitment, illuminating a regulatory role for ribosome levels in cellular differentiation.
Assuntos
Anemia de Diamond-Blackfan/patologia , Ribossomos/metabolismo , Regiões 5' não Traduzidas , Anemia de Diamond-Blackfan/genética , Proteínas Reguladoras de Apoptose/antagonistas & inibidores , Proteínas Reguladoras de Apoptose/genética , Proteínas Reguladoras de Apoptose/metabolismo , Células da Medula Óssea/metabolismo , Células Cultivadas , Feminino , Fator de Transcrição GATA1/genética , Fator de Transcrição GATA1/metabolismo , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/metabolismo , Humanos , Masculino , Mutação de Sentido Incorreto , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Proteínas Ribossômicas/antagonistas & inibidores , Proteínas Ribossômicas/genética , Proteínas Ribossômicas/metabolismo , Ribossomos/genética , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
RNA profiles are an informative phenotype of cellular and tissue states but can be costly to generate at massive scale. Here, we describe how gene expression levels can be efficiently acquired with random composite measurements-in which abundances are combined in a random weighted sum. We show (1) that the similarity between pairs of expression profiles can be approximated with very few composite measurements; (2) that by leveraging sparse, modular representations of gene expression, we can use random composite measurements to recover high-dimensional gene expression levels (with 100 times fewer measurements than genes); and (3) that it is possible to blindly recover gene expression from composite measurements, even without access to training data. Our results suggest new compressive modalities as a foundation for massive scaling in high-throughput measurements and new insights into the interpretation of high-dimensional data.
Assuntos
Algoritmos , Perfilação da Expressão Gênica/métodos , Compressão de Dados , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Humanos , Células K562 , Análise de Sequência de RNA/métodosRESUMO
The genetic dependencies of human cancers widely vary. Here, we catalog this heterogeneity and use it to identify functional gene interactions and genotype-dependent liabilities in cancer. By using genome-wide CRISPR-based screens, we generate a gene essentiality dataset across 14 human acute myeloid leukemia (AML) cell lines. Sets of genes with correlated patterns of essentiality across the lines reveal new gene relationships, the essential substrates of enzymes, and the molecular functions of uncharacterized proteins. Comparisons of differentially essential genes between Ras-dependent and -independent lines uncover synthetic lethal partners of oncogenic Ras. Screens in both human AML and engineered mouse pro-B cells converge on a surprisingly small number of genes in the Ras processing and MAPK pathways and pinpoint PREX1 as an AML-specific activator of MAPK signaling. Our findings suggest general strategies for defining mammalian gene networks and synthetic lethal interactions by exploiting the natural genetic and epigenetic diversity of human cancer cells.
Assuntos
Redes Reguladoras de Genes , Leucemia Mieloide Aguda/genética , Animais , Proteínas de Transporte , Linhagem Celular Tumoral , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Epigênese Genética , Genes Essenciais , Humanos , Sistema de Sinalização das MAP Quinases , Camundongos , Proteínas Mitocondriais , Processamento de Proteína Pós-Traducional , Proteínas ras/genéticaRESUMO
The human genome folds to create thousands of intervals, called "contact domains," that exhibit enhanced contact frequency within themselves. "Loop domains" form because of tethering between two loci-almost always bound by CTCF and cohesin-lying on the same chromosome. "Compartment domains" form when genomic intervals with similar histone marks co-segregate. Here, we explore the effects of degrading cohesin. All loop domains are eliminated, but neither compartment domains nor histone marks are affected. Loss of loop domains does not lead to widespread ectopic gene activation but does affect a significant minority of active genes. In particular, cohesin loss causes superenhancers to co-localize, forming hundreds of links within and across chromosomes and affecting the regulation of nearby genes. We then restore cohesin and monitor the re-formation of each loop. Although re-formation rates vary greatly, many megabase-sized loops recovered in under an hour, consistent with a model where loop extrusion is rapid.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Núcleo Celular/genética , Proteínas Cromossômicas não Histona/metabolismo , Cromossomos/metabolismo , Genoma Humano , Proteínas Repressoras/metabolismo , Fator de Ligação a CCCTC , Linhagem Celular Tumoral , Proteínas de Ligação a DNA , Elementos Facilitadores Genéticos , Código das Histonas , Humanos , Proteínas Nucleares/metabolismo , Nucleossomos/metabolismo , Fosfoproteínas/metabolismo , CoesinasRESUMO
Cytokines are classically thought to stimulate downstream signaling pathways through monotonic activation of receptors. We describe a severe anemia resulting from a homozygous mutation (R150Q) in the cytokine erythropoietin (EPO). Surprisingly, the EPO R150Q mutant shows only a mild reduction in affinity for its receptor but has altered binding kinetics. The EPO mutant is less effective at stimulating erythroid cell proliferation and differentiation, even at maximally potent concentrations. While the EPO mutant can stimulate effectors such as STAT5 to a similar extent as the wild-type ligand, there is reduced JAK2-mediated phosphorylation of select downstream targets. This impairment in downstream signaling mechanistically arises from altered receptor dimerization dynamics due to extracellular binding changes. These results demonstrate how variation in a single cytokine can lead to biased downstream signaling and can thereby cause human disease. Moreover, we have defined a distinct treatable form of anemia through mutation identification and functional studies.
Assuntos
Anemia de Diamond-Blackfan/genética , Anemia de Diamond-Blackfan/patologia , Eritropoetina/genética , Mutação de Sentido Incorreto , Transdução de Sinais , Anemia de Diamond-Blackfan/terapia , Criança , Consanguinidade , Ativação Enzimática , Eritropoese , Eritropoetina/química , Feminino , Humanos , Janus Quinase 2/metabolismo , Cinética , Masculino , Receptores da Eritropoetina/química , Receptores da Eritropoetina/genética , Receptores da Eritropoetina/metabolismoRESUMO
Type 2 diabetes (T2D) affects Latinos at twice the rate seen in populations of European descent. We recently identified a risk haplotype spanning SLC16A11 that explains â¼20% of the increased T2D prevalence in Mexico. Here, through genetic fine-mapping, we define a set of tightly linked variants likely to contain the causal allele(s). We show that variants on the T2D-associated haplotype have two distinct effects: (1) decreasing SLC16A11 expression in liver and (2) disrupting a key interaction with basigin, thereby reducing cell-surface localization. Both independent mechanisms reduce SLC16A11 function and suggest SLC16A11 is the causal gene at this locus. To gain insight into how SLC16A11 disruption impacts T2D risk, we demonstrate that SLC16A11 is a proton-coupled monocarboxylate transporter and that genetic perturbation of SLC16A11 induces changes in fatty acid and lipid metabolism that are associated with increased T2D risk. Our findings suggest that increasing SLC16A11 function could be therapeutically beneficial for T2D. VIDEO ABSTRACT.
Assuntos
Diabetes Mellitus Tipo 2/metabolismo , Transportadores de Ácidos Monocarboxílicos/genética , Transportadores de Ácidos Monocarboxílicos/metabolismo , Basigina/metabolismo , Membrana Celular/metabolismo , Cromossomos Humanos Par 17/metabolismo , Técnicas de Silenciamento de Genes , Haplótipos , Hepatócitos/metabolismo , Heterozigoto , Código das Histonas , Humanos , Fígado/metabolismo , Modelos Moleculares , Transportadores de Ácidos Monocarboxílicos/químicaRESUMO
Three years ago, scientists reported that CRISPR technology can enable precise and efficient genome editing in living eukaryotic cells. Since then, the method has taken the scientific community by storm, with thousands of labs using it for applications from biomedicine to agriculture. Yet, the preceding 20-year journey--the discovery of a strange microbial repeat sequence; its recognition as an adaptive immune system; its biological characterization; and its repurposing for genome engineering--remains little known. This Perspective aims to fill in this backstory--the history of ideas and the stories of pioneers--and draw lessons about the remarkable ecosystem underlying scientific discovery.
Assuntos
Sistemas CRISPR-Cas , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Engenharia Genética/história , Engenharia Genética/métodos , Pessoal de Laboratório , Imunidade Adaptativa , Animais , Archaea/classificação , Archaea/genética , Archaea/imunologia , Archaea/virologia , Bactérias/classificação , Bactérias/genética , Bactérias/imunologia , Bactérias/virologia , Pesquisa Biomédica , Haloferax mediterranei/genética , Haloferax mediterranei/imunologia , História do Século XX , História do Século XXI , HumanosRESUMO
Although studies have identified hundreds of loci associated with human traits and diseases, pinpointing causal alleles remains difficult, particularly for non-coding variants. To address this challenge, we adapted the massively parallel reporter assay (MPRA) to identify variants that directly modulate gene expression. We applied it to 32,373 variants from 3,642 cis-expression quantitative trait loci and control regions. Detection by MPRA was strongly correlated with measures of regulatory function. We demonstrate MPRA's capabilities for pinpointing causal alleles, using it to identify 842 variants showing differential expression between alleles, including 53 well-annotated variants associated with diseases and traits. We investigated one in detail, a risk allele for ankylosing spondylitis, and provide direct evidence of a non-coding variant that alters expression of the prostaglandin EP4 receptor. These results create a resource of concrete leads and illustrate the promise of this approach for comprehensively interrogating how non-coding polymorphism shapes human biology.
Assuntos
Regulação da Expressão Gênica , Genes Reporter , Doenças Genéticas Inatas/genética , Técnicas Genéticas , Variação Genética , Alelos , Biblioteca Gênica , Células Hep G2 , Humanos , Locos de Características Quantitativas , Sensibilidade e Especificidade , Espondilite Anquilosante/genéticaRESUMO
Genetic screens help infer gene function in mammalian cells, but it has remained difficult to assay complex phenotypes-such as transcriptional profiles-at scale. Here, we develop Perturb-seq, combining single-cell RNA sequencing (RNA-seq) and clustered regularly interspaced short palindromic repeats (CRISPR)-based perturbations to perform many such assays in a pool. We demonstrate Perturb-seq by analyzing 200,000 cells in immune cells and cell lines, focusing on transcription factors regulating the response of dendritic cells to lipopolysaccharide (LPS). Perturb-seq accurately identifies individual gene targets, gene signatures, and cell states affected by individual perturbations and their genetic interactions. We posit new functions for regulators of differentiation, the anti-viral response, and mitochondrial function during immune activation. By decomposing many high content measurements into the effects of perturbations, their interactions, and diverse cell metadata, Perturb-seq dramatically increases the scope of pooled genomic assays.
Assuntos
Análise de Sequência de RNA/métodos , Análise de Célula Única/métodos , Animais , Ciclo Celular , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Retroalimentação , Perfilação da Expressão Gênica , Técnicas de Silenciamento de Genes , Humanos , Células K562 , Camundongos , Camundongos Transgênicos , Fatores de Transcrição/metabolismoRESUMO
Induced pluripotency is a promising avenue for disease modeling and therapy, but the molecular principles underlying this process, particularly in human cells, remain poorly understood due to donor-to-donor variability and intercellular heterogeneity. Here, we constructed and characterized a clonal, inducible human reprogramming system that provides a reliable source of cells at any stage of the process. This system enabled integrative transcriptional and epigenomic analysis across the human reprogramming timeline at high resolution. We observed distinct waves of gene network activation, including the ordered re-activation of broad developmental regulators followed by early embryonic patterning genes and culminating in the emergence of a signature reminiscent of pre-implantation stages. Moreover, complementary functional analyses allowed us to identify and validate novel regulators of the reprogramming process. Altogether, this study sheds light on the molecular underpinnings of induced pluripotency in human cells and provides a robust cell platform for further studies. PAPERCLIP.
Assuntos
Reprogramação Celular , Células-Tronco Pluripotentes Induzidas/citologia , Cromatina/metabolismo , Montagem e Desmontagem da Cromatina , Epigênese Genética , Perfilação da Expressão Gênica , Histona Desmetilases/metabolismo , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismoRESUMO
Linking variants from genome-wide association studies (GWAS) to underlying mechanisms of disease remains a challenge1-3. For some diseases, a successful strategy has been to look for cases in which multiple GWAS loci contain genes that act in the same biological pathway1-6. However, our knowledge of which genes act in which pathways is incomplete, particularly for cell-type-specific pathways or understudied genes. Here we introduce a method to connect GWAS variants to functions. This method links variants to genes using epigenomics data, links genes to pathways de novo using Perturb-seq and integrates these data to identify convergence of GWAS loci onto pathways. We apply this approach to study the role of endothelial cells in genetic risk for coronary artery disease (CAD), and discover 43 CAD GWAS signals that converge on the cerebral cavernous malformation (CCM) signalling pathway. Two regulators of this pathway, CCM2 and TLNRD1, are each linked to a CAD risk variant, regulate other CAD risk genes and affect atheroprotective processes in endothelial cells. These results suggest a model whereby CAD risk is driven in part by the convergence of causal genes onto a particular transcriptional pathway in endothelial cells. They highlight shared genes between common and rare vascular diseases (CAD and CCM), and identify TLNRD1 as a new, previously uncharacterized member of the CCM signalling pathway. This approach will be widely useful for linking variants to functions for other common polygenic diseases.
Assuntos
Doença da Artéria Coronariana , Células Endoteliais , Estudo de Associação Genômica Ampla , Hemangioma Cavernoso do Sistema Nervoso Central , Humanos , Doença da Artéria Coronariana/genética , Doença da Artéria Coronariana/patologia , Células Endoteliais/metabolismo , Células Endoteliais/patologia , Predisposição Genética para Doença/genética , Hemangioma Cavernoso do Sistema Nervoso Central/genética , Hemangioma Cavernoso do Sistema Nervoso Central/patologia , Polimorfismo de Nucleotídeo Único , Epigenômica , Transdução de Sinais/genética , Herança MultifatorialRESUMO
Recent advances in genome engineering technologies based on the CRISPR-associated RNA-guided endonuclease Cas9 are enabling the systematic interrogation of mammalian genome function. Analogous to the search function in modern word processors, Cas9 can be guided to specific locations within complex genomes by a short RNA search string. Using this system, DNA sequences within the endogenous genome and their functional outputs are now easily edited or modulated in virtually any organism of choice. Cas9-mediated genetic perturbation is simple and scalable, empowering researchers to elucidate the functional organization of the genome at the systems level and establish causal linkages between genetic variations and biological phenotypes. In this Review, we describe the development and applications of Cas9 for a variety of research or translational applications while highlighting challenges as well as future directions. Derived from a remarkable microbial defense system, Cas9 is driving innovative applications from basic biology to biotechnology and medicine.
Assuntos
Bactérias/genética , Sistemas CRISPR-Cas , Marcação de Genes , Engenharia Genética , Animais , Bactérias/classificação , Bactérias/imunologia , Bactérias/virologia , Células Eucarióticas/metabolismo , Genoma , Humanos , Streptococcus pyogenes/enzimologia , Streptococcus pyogenes/genéticaRESUMO
Intermolecular RNA-RNA interactions are used by many noncoding RNAs (ncRNAs) to achieve their diverse functions. To identify these contacts, we developed a method based on RNA antisense purification to systematically map RNA-RNA interactions (RAP-RNA) and applied it to investigate two ncRNAs implicated in RNA processing: U1 small nuclear RNA, a component of the spliceosome, and Malat1, a large ncRNA that localizes to nuclear speckles. U1 and Malat1 interact with nascent transcripts through distinct targeting mechanisms. Using differential crosslinking, we confirmed that U1 directly hybridizes to 5' splice sites and 5' splice site motifs throughout introns and found that Malat1 interacts with pre-mRNAs indirectly through protein intermediates. Interactions with nascent pre-mRNAs cause U1 and Malat1 to localize proximally to chromatin at active genes, demonstrating that ncRNAs can use RNA-RNA interactions to target specific pre-mRNAs and genomic sites. RAP-RNA is sensitive to lower abundance RNAs as well, making it generally applicable for investigating ncRNAs.
Assuntos
Técnicas Genéticas , RNA Mensageiro/metabolismo , Animais , Sequência de Bases , Reagentes de Ligações Cruzadas/metabolismo , Camundongos , Dados de Sequência Molecular , Motivos de Nucleotídeos , Sítios de Splice de RNA , RNA Longo não Codificante/química , RNA Longo não Codificante/metabolismo , RNA Mensageiro/química , RNA Nuclear Pequeno/metabolismo , RNA não Traduzido/química , RNA não Traduzido/metabolismoRESUMO
We use in situ Hi-C to probe the 3D architecture of genomes, constructing haploid and diploid maps of nine cell types. The densest, in human lymphoblastoid cells, contains 4.9 billion contacts, achieving 1 kb resolution. We find that genomes are partitioned into contact domains (median length, 185 kb), which are associated with distinct patterns of histone marks and segregate into six subcompartments. We identify â¼10,000 loops. These loops frequently link promoters and enhancers, correlate with gene activation, and show conservation across cell types and species. Loop anchors typically occur at domain boundaries and bind CTCF. CTCF sites at loop anchors occur predominantly (>90%) in a convergent orientation, with the asymmetric motifs "facing" one another. The inactive X chromosome splits into two massive domains and contains large loops anchored at CTCF-binding repeats.