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1.
Cell ; 156(5): 935-49, 2014 Feb 27.
Artigo em Inglês | MEDLINE | ID: mdl-24529477

RESUMO

The CRISPR-associated endonuclease Cas9 can be targeted to specific genomic loci by single guide RNAs (sgRNAs). Here, we report the crystal structure of Streptococcus pyogenes Cas9 in complex with sgRNA and its target DNA at 2.5 Å resolution. The structure revealed a bilobed architecture composed of target recognition and nuclease lobes, accommodating the sgRNA:DNA heteroduplex in a positively charged groove at their interface. Whereas the recognition lobe is essential for binding sgRNA and DNA, the nuclease lobe contains the HNH and RuvC nuclease domains, which are properly positioned for cleavage of the complementary and noncomplementary strands of the target DNA, respectively. The nuclease lobe also contains a carboxyl-terminal domain responsible for the interaction with the protospacer adjacent motif (PAM). This high-resolution structure and accompanying functional analyses have revealed the molecular mechanism of RNA-guided DNA targeting by Cas9, thus paving the way for the rational design of new, versatile genome-editing technologies.


Assuntos
Proteínas Associadas a CRISPR/química , Cristalografia por Raios X , Endonucleases/química , RNA Bacteriano/química , Streptococcus pyogenes/química , Sequência de Aminoácidos , Bactérias/enzimologia , Proteínas Associadas a CRISPR/metabolismo , DNA Bacteriano/química , DNA Bacteriano/metabolismo , Endonucleases/metabolismo , Modelos Moleculares , Dados de Sequência Molecular , Estrutura Terciária de Proteína , RNA Bacteriano/metabolismo , Alinhamento de Sequência , Streptococcus pyogenes/enzimologia , Streptococcus pyogenes/metabolismo , Pequeno RNA não Traduzido
2.
Nature ; 573(7772): 61-68, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31435019

RESUMO

Elucidating the cellular architecture of the human cerebral cortex is central to understanding our cognitive abilities and susceptibility to disease. Here we used single-nucleus RNA-sequencing analysis to perform a comprehensive study of cell types in the middle temporal gyrus of human cortex. We identified a highly diverse set of excitatory and inhibitory neuron types that are mostly sparse, with excitatory types being less layer-restricted than expected. Comparison to similar mouse cortex single-cell RNA-sequencing datasets revealed a surprisingly well-conserved cellular architecture that enables matching of homologous types and predictions of properties of human cell types. Despite this general conservation, we also found extensive differences between homologous human and mouse cell types, including marked alterations in proportions, laminar distributions, gene expression and morphology. These species-specific features emphasize the importance of directly studying human brain.


Assuntos
Astrócitos/classificação , Evolução Biológica , Córtex Cerebral/citologia , Córtex Cerebral/metabolismo , Neurônios/classificação , Adolescente , Adulto , Idoso , Animais , Astrócitos/citologia , Feminino , Humanos , Masculino , Camundongos , Pessoa de Meia-Idade , Inibição Neural , Neurônios/citologia , Análise de Componente Principal , RNA-Seq , Análise de Célula Única , Especificidade da Espécie , Transcriptoma/genética , Adulto Jovem
3.
Virol J ; 21(1): 38, 2024 02 06.
Artigo em Inglês | MEDLINE | ID: mdl-38321453

RESUMO

During viral infection there is dynamic interplay between the virus and the host to regulate gene expression. In many cases, the host induces the expression of antiviral genes to combat infection, while the virus uses "host shut-off" systems to better compete for cellular resources and to limit the induction of the host antiviral response. Viral mechanisms for host shut-off involve targeting translation, altering host RNA processing, and/or inducing the degradation of host mRNAs. In this review, we discuss the diverse mechanisms viruses use to degrade host mRNAs. In addition, the widespread degradation of host mRNAs can have common consequences including the accumulation of RNA binding proteins in the nucleus, which leads to altered RNA processing, mRNA export, and changes to transcription.


Assuntos
Viroses , Vírus , Humanos , Regulação da Expressão Gênica , RNA Mensageiro/genética , Vírus/genética , Antivirais , Replicação Viral
4.
RNA Biol ; 20(1): 444-456, 2023 01.
Artigo em Inglês | MEDLINE | ID: mdl-37415298

RESUMO

Nsp1 is a SARS-CoV-2 host shutoff factor that both represses cellular translation and promotes host RNA decay. However, it is unclear how these two activities are connected and interact with normal translation processes. Here, we performed mutational analyses of Nsp1, and these revealed that both the N and C terminal domains of Nsp1 are important for translational repression. Furthermore, we demonstrate that specific residues in the N terminal domain are required for cellular RNA degradation but not bulk translation shutoff of host mRNAs, thereby separating RNA degradation from translation repression. We also present evidence that Nsp1 mediated RNA degradation requires engagement of the ribosome with mRNA. First, we observe that cytosolic lncRNAs, which are not translated, escape Nsp1 mediated degradation. Second, inhibition of translation elongation with emetine does not prevent Nsp1 mediated degradation, while blocking translation initiation before 48S ribosome loading reduces mRNA degradation. Taken together, we suggest that Nsp1 represses translation and promotes mRNA degradation only after ribosome engagement with the mRNA. This raises the possibility that Nsp1 may trigger RNA degradation through pathways that recognize stalled ribosomes.


Assuntos
COVID-19 , SARS-CoV-2 , Humanos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , SARS-CoV-2/genética , SARS-CoV-2/metabolismo , Proteínas não Estruturais Virais/metabolismo , COVID-19/metabolismo , Biossíntese de Proteínas , Ribossomos/metabolismo , Estabilidade de RNA
5.
Nat Methods ; 13(1): 87-93, 2016 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-26524239

RESUMO

The diverse progenitors that give rise to the human neocortex have been difficult to characterize because progenitors, particularly radial glia (RG), are rare and are defined by a combination of intracellular markers, position and morphology. To circumvent these problems, we developed Fixed and Recovered Intact Single-cell RNA (FRISCR), a method for profiling the transcriptomes of individual fixed, stained and sorted cells. Using FRISCR, we profiled primary human RG that constitute only 1% of the midgestation cortex and classified them as ventricular zone-enriched RG (vRG) that express ANXA1 and CRYAB, and outer subventricular zone-localized RG (oRG) that express HOPX. Our study identified vRG and oRG markers and molecular profiles, an essential step for understanding human neocortical progenitor development. FRISCR allows targeted single-cell profiling of any tissues that lack live-cell markers.


Assuntos
Encéfalo/citologia , Neuroglia/citologia , Transcriptoma , Humanos , Análise de Célula Única
6.
Science ; 382(6667): eadf6812, 2023 10 13.
Artigo em Inglês | MEDLINE | ID: mdl-37824655

RESUMO

Variation in cytoarchitecture is the basis for the histological definition of cortical areas. We used single cell transcriptomics and performed cellular characterization of the human cortex to better understand cortical areal specialization. Single-nucleus RNA-sequencing of 8 areas spanning cortical structural variation showed a highly consistent cellular makeup for 24 cell subclasses. However, proportions of excitatory neuron subclasses varied substantially, likely reflecting differences in connectivity across primary sensorimotor and association cortices. Laminar organization of astrocytes and oligodendrocytes also differed across areas. Primary visual cortex showed characteristic organization with major changes in the excitatory to inhibitory neuron ratio, expansion of layer 4 excitatory neurons, and specialized inhibitory neurons. These results lay the groundwork for a refined cellular and molecular characterization of human cortical cytoarchitecture and areal specialization.


Assuntos
Neocórtex , Humanos , Neocórtex/metabolismo , Neocórtex/ultraestrutura , Neurônios/classificação , Neurônios/metabolismo , Transcriptoma , Análise da Expressão Gênica de Célula Única , Filogenia
7.
Sci Adv ; 7(1)2021 01.
Artigo em Inglês | MEDLINE | ID: mdl-33219112

RESUMO

The COVID-19 pandemic has created a public health crisis. Because SARS-CoV-2 can spread from individuals with presymptomatic, symptomatic, and asymptomatic infections, the reopening of societies and the control of virus spread will be facilitated by robust population screening, for which virus testing will often be central. After infection, individuals undergo a period of incubation during which viral titers are too low to detect, followed by exponential viral growth, leading to peak viral load and infectiousness and ending with declining titers and clearance. Given the pattern of viral load kinetics, we model the effectiveness of repeated population screening considering test sensitivities, frequency, and sample-to-answer reporting time. These results demonstrate that effective screening depends largely on frequency of testing and speed of reporting and is only marginally improved by high test sensitivity. We therefore conclude that screening should prioritize accessibility, frequency, and sample-to-answer time; analytical limits of detection should be secondary.


Assuntos
Teste de Ácido Nucleico para COVID-19 , COVID-19/diagnóstico , Programas de Rastreamento/métodos , Carga Viral , Infecções Assintomáticas , Calibragem , Simulação por Computador , Epidemias , Humanos , Cinética , Limite de Detecção , Modelos Teóricos , Reação em Cadeia da Polimerase , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Fatores de Tempo
8.
HIV Res Clin Pract ; 22(4): 102-118, 2021 08.
Artigo em Inglês | MEDLINE | ID: mdl-34514963

RESUMO

Background:Understanding the relationship between HIV and SARS-CoV-2 has important public health implications.Objective:To summarize current research on COVID-19 among people with HIV (PWH) as published through 15 July 2021.Methods: We conducted a search of PubMed, Scopus, preprint databases (medRxiv, bioRxiv), and the references of publications found using key terms relevant to COVID-19 ('COVID-19' OR 'SARS-CoV-2' OR 'coronavirus') AND to HIV ('HIV' OR 'Human Immunodeficiency Virus' OR 'AIDS' OR 'Acquired Immunodeficiency Syndrome'). We summarized all articles that reported data or opinions on SARS-CoV-2 and HIV coinfection.Conclusions: Although many initial case series and cohort studies found no increased risk for SARS-CoV-2 infection or severe COVID-19 outcomes among PWH, recent studies have signaled an increased risk for severe COVID-19 disease progression even in the setting of well-controlled HIV. Whether this is due to the increased prevalence of comorbidities in PWH and other social determinants of health is unknown. These conflicting findings highlight the continued need for COVID-19 related research among PWH that addresses COVID-19 disease course as well as exacerbation of existing comorbidities already disproportionately represented among PWH.


Assuntos
COVID-19/complicações , Infecções por HIV/complicações , Animais , COVID-19/epidemiologia , COVID-19/virologia , Coinfecção/epidemiologia , Coinfecção/virologia , Infecções por HIV/epidemiologia , Infecções por HIV/virologia , HIV-1/genética , HIV-1/fisiologia , Humanos , SARS-CoV-2/genética , SARS-CoV-2/fisiologia
9.
PLoS One ; 16(6): e0237055, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34166368

RESUMO

A key aspect in defining cell state is the complex choreography of DNA binding events in a given cell type, which in turn establishes a cell-specific gene-expression program. Here we wanted to take a deep analysis of DNA binding events and transcriptional output of a single cell state (K562 cells). To this end we re-analyzed 195 DNA binding proteins contained in ENCODE data. We used standardized analysis pipelines, containerization, and literate programming with R Markdown for reproducibility and rigor. Our approach validated many findings from previous independent studies, underscoring the importance of ENCODE's goals in providing these reproducible data resources. We also had several new findings including: (i) 1,362 promoters, which we refer to as 'reservoirs,' that are defined by having up to 111 different DNA binding-proteins localized on one promoter, yet do not have any expression of steady-state RNA (ii) Reservoirs do not overlap super-enhancer annotations and distinct have distinct properties from super-enhancers. (iii) The human specific SVA repeat element may have been co-opted for enhancer regulation and is highly transcribed in PRO-seq and RNA-seq. Collectively, this study performed by the students of a CU Boulder computational biology class (BCHM 5631 -Spring 2020) demonstrates the value of reproducible findings and how resources like ENCODE that prioritize data standards can foster new findings with existing data in a didactic environment.


Assuntos
Biologia Computacional/métodos , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Regulação da Expressão Gênica , Genoma Humano , Regiões Promotoras Genéticas , Sequências Repetitivas de Ácido Nucleico , Humanos , Células K562 , Reprodutibilidade dos Testes
10.
Elife ; 102021 09 02.
Artigo em Inglês | MEDLINE | ID: mdl-34473054

RESUMO

Abundant evidence supports the presence of at least three distinct types of thalamocortical (TC) neurons in the primate dorsal lateral geniculate nucleus (dLGN) of the thalamus, the brain region that conveys visual information from the retina to the primary visual cortex (V1). Different types of TC neurons in mice, humans, and macaques have distinct morphologies, distinct connectivity patterns, and convey different aspects of visual information to the cortex. To investigate the molecular underpinnings of these cell types, and how these relate to differences in dLGN between human, macaque, and mice, we profiled gene expression in single nuclei and cells using RNA-sequencing. These efforts identified four distinct types of TC neurons in the primate dLGN: magnocellular (M) neurons, parvocellular (P) neurons, and two types of koniocellular (K) neurons. Despite extensively documented morphological and physiological differences between M and P neurons, we identified few genes with significant differential expression between transcriptomic cell types corresponding to these two neuronal populations. Likewise, the dominant feature of TC neurons of the adult mouse dLGN is high transcriptomic similarity, with an axis of heterogeneity that aligns with core vs. shell portions of mouse dLGN. Together, these data show that transcriptomic differences between principal cell types in the mature mammalian dLGN are subtle relative to the observed differences in morphology and cortical projection targets. Finally, alignment of transcriptome profiles across species highlights expanded diversity of GABAergic neurons in primate versus mouse dLGN and homologous types of TC neurons in primates that are distinct from TC neurons in mouse.


Assuntos
Núcleo Celular/genética , Corpos Geniculados/metabolismo , Neurônios/metabolismo , Córtex Visual/metabolismo , Animais , Perfilação da Expressão Gênica , Humanos , Macaca , Camundongos , RNA-Seq , Análise de Célula Única , Tálamo/metabolismo , Vias Visuais/metabolismo
11.
medRxiv ; 2020 Sep 08.
Artigo em Inglês | MEDLINE | ID: mdl-32607516

RESUMO

The COVID-19 pandemic has created a public health crisis. Because SARS-CoV-2 can spread from individuals with pre-symptomatic, symptomatic, and asymptomatic infections, the re-opening of societies and the control of virus spread will be facilitated by robust surveillance, for which virus testing will often be central. After infection, individuals undergo a period of incubation during which viral titers are usually too low to detect, followed by an exponential viral growth, leading to a peak viral load and infectiousness, and ending with declining viral levels and clearance. Given the pattern of viral load kinetics, we model surveillance effectiveness considering test sensitivities, frequency, and sample-to-answer reporting time. These results demonstrate that effective surveillance depends largely on frequency of testing and the speed of reporting, and is only marginally improved by high test sensitivity. We therefore conclude that surveillance should prioritize accessibility, frequency, and sample-to-answer time; analytical limits of detection should be secondary.

12.
Nat Commun ; 11(1): 1172, 2020 03 03.
Artigo em Inglês | MEDLINE | ID: mdl-32127543

RESUMO

von Economo neurons (VENs) are bipolar, spindle-shaped neurons restricted to layer 5 of human frontoinsula and anterior cingulate cortex that appear to be selectively vulnerable to neuropsychiatric and neurodegenerative diseases, although little is known about other VEN cellular phenotypes. Single nucleus RNA-sequencing of frontoinsula layer 5 identifies a transcriptomically-defined cell cluster that contained VENs, but also fork cells and a subset of pyramidal neurons. Cross-species alignment of this cell cluster with a well-annotated mouse classification shows strong homology to extratelencephalic (ET) excitatory neurons that project to subcerebral targets. This cluster also shows strong homology to a putative ET cluster in human temporal cortex, but with a strikingly specific regional signature. Together these results suggest that VENs are a regionally distinctive type of ET neuron. Additionally, we describe the first patch clamp recordings of VENs from neurosurgically-resected tissue that show distinctive intrinsic membrane properties relative to neighboring pyramidal neurons.


Assuntos
Neurônios/fisiologia , Lobo Temporal/citologia , Transcriptoma , Animais , Encéfalo/citologia , Encéfalo/fisiologia , Eletrofisiologia/métodos , Perfilação da Expressão Gênica , Humanos , Hibridização in Situ Fluorescente , Camundongos , Neurônios/citologia , Células Piramidais/fisiologia , Telencéfalo/citologia , Lobo Temporal/fisiologia
13.
PLoS One ; 13(12): e0209648, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30586455

RESUMO

Transcriptomic profiling of complex tissues by single-nucleus RNA-sequencing (snRNA-seq) affords some advantages over single-cell RNA-sequencing (scRNA-seq). snRNA-seq provides less biased cellular coverage, does not appear to suffer cell isolation-based transcriptional artifacts, and can be applied to archived frozen specimens. We used well-matched snRNA-seq and scRNA-seq datasets from mouse visual cortex to compare cell type detection. Although more transcripts are detected in individual whole cells (~11,000 genes) than nuclei (~7,000 genes), we demonstrate that closely related neuronal cell types can be similarly discriminated with both methods if intronic sequences are included in snRNA-seq analysis. We estimate that the nuclear proportion of total cellular mRNA varies from 20% to over 50% for large and small pyramidal neurons, respectively. Together, these results illustrate the high information content of nuclear RNA for characterization of cellular diversity in brain tissues.


Assuntos
Núcleo Celular/genética , Análise de Célula Única , Transcriptoma/genética , Córtex Visual/metabolismo , Animais , Linhagem da Célula/genética , Linhagem da Célula/fisiologia , Perfilação da Expressão Gênica/métodos , Camundongos , Neurônios/metabolismo , Análise de Sequência de RNA/métodos , Córtex Visual/fisiologia
14.
Nat Neurosci ; 21(9): 1185-1195, 2018 09.
Artigo em Inglês | MEDLINE | ID: mdl-30150662

RESUMO

We describe convergent evidence from transcriptomics, morphology, and physiology for a specialized GABAergic neuron subtype in human cortex. Using unbiased single-nucleus RNA sequencing, we identify ten GABAergic interneuron subtypes with combinatorial gene signatures in human cortical layer 1 and characterize a group of human interneurons with anatomical features never described in rodents, having large 'rosehip'-like axonal boutons and compact arborization. These rosehip cells show an immunohistochemical profile (GAD1+CCK+, CNR1-SST-CALB2-PVALB-) matching a single transcriptomically defined cell type whose specific molecular marker signature is not seen in mouse cortex. Rosehip cells in layer 1 make homotypic gap junctions, predominantly target apical dendritic shafts of layer 3 pyramidal neurons, and inhibit backpropagating pyramidal action potentials in microdomains of the dendritic tuft. These cells are therefore positioned for potent local control of distal dendritic computation in cortical pyramidal neurons.


Assuntos
Córtex Cerebral/metabolismo , Córtex Cerebral/ultraestrutura , Neurônios GABAérgicos/metabolismo , Neurônios GABAérgicos/ultraestrutura , Transcriptoma , Adulto , Idoso , Axônios/ultraestrutura , Espinhas Dendríticas/metabolismo , Espinhas Dendríticas/ultraestrutura , Junções Comunicantes/metabolismo , Junções Comunicantes/ultraestrutura , Biblioteca Gênica , Humanos , Masculino , Reação em Cadeia da Polimerase , Terminações Pré-Sinápticas/metabolismo , Terminações Pré-Sinápticas/ultraestrutura , Células Piramidais/metabolismo , Células Piramidais/ultraestrutura , RNA/análise , RNA/genética , Análise de Sequência de RNA
15.
Neuron ; 93(5): 1035-1048.e5, 2017 Mar 08.
Artigo em Inglês | MEDLINE | ID: mdl-28279351

RESUMO

GABAergic interneurons are essential for neural circuit function, and their loss or dysfunction is implicated in human neuropsychiatric disease. In vitro methods for interneuron generation hold promise for studying human cellular and functional properties and, ultimately, for therapeutic cell replacement. Here we describe a protocol for generating cortical interneurons from hESCs and analyze the properties and maturation time course of cell types using single-cell RNA-seq. We find that the cell types produced mimic in vivo temporal patterns of neuron and glial production, with immature progenitors and neurons observed early and mature cortical neurons and glial cell types produced late. By comparing the transcriptomes of immature interneurons to those of more mature neurons, we identified genes important for human interneuron differentiation. Many of these genes were previously implicated in neurodevelopmental and neuropsychiatric disorders.


Assuntos
Diferenciação Celular/fisiologia , Movimento Celular/fisiologia , Neurônios GABAérgicos/citologia , Interneurônios/citologia , Proteínas do Tecido Nervoso/metabolismo , Neuroglia/citologia , Células Cultivadas , Humanos , Neurogênese/fisiologia , Análise de Célula Única , Fatores de Transcrição/metabolismo
16.
Cell Stem Cell ; 20(1): 120-134, 2017 01 05.
Artigo em Inglês | MEDLINE | ID: mdl-28094016

RESUMO

During human brain development, multiple signaling pathways generate diverse cell types with varied regional identities. Here, we integrate single-cell RNA sequencing and clonal analyses to reveal lineage trees and molecular signals underlying early forebrain and mid/hindbrain cell differentiation from human embryonic stem cells (hESCs). Clustering single-cell transcriptomic data identified 41 distinct populations of progenitor, neuronal, and non-neural cells across our differentiation time course. Comparisons with primary mouse and human gene expression data demonstrated rostral and caudal progenitor and neuronal identities from early brain development. Bayesian analyses inferred a unified cell-type lineage tree that bifurcates between cortical and mid/hindbrain cell types. Two methods of clonal analyses confirmed these findings and further revealed the importance of Wnt/ß-catenin signaling in controlling this lineage decision. Together, these findings provide a rich transcriptome-based lineage map for studying human brain development and modeling developmental disorders.


Assuntos
Encéfalo/embriologia , Linhagem da Célula , Desenvolvimento Embrionário , Células-Tronco Embrionárias Humanas/citologia , Análise de Célula Única/métodos , Animais , Encéfalo/metabolismo , Linhagem Celular , Linhagem da Célula/genética , Células Clonais , Desenvolvimento Embrionário/genética , Humanos , Camundongos , Modelos Biológicos , Neurônios/citologia , Neurônios/metabolismo , Reprodutibilidade dos Testes , Análise de Sequência de RNA , Fatores de Transcrição/metabolismo , Transcriptoma/genética , Via de Sinalização Wnt/genética
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