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1.
Zhongguo Shi Yan Xue Ye Xue Za Zhi ; 29(2): 339-347, 2021 Apr.
Artículo en Chino | MEDLINE | ID: mdl-33812397

RESUMEN

OBJECTIVE: To identify differentiation related miRNA and evaluate roles of miRNA during ATRA induced myeloid differentiation. METHODS: The small RNA sequencing was used to analyze differential expressed miRNAs in ATRA induced NB4 cells. Then the several up or down-regulated miRNA were selected as the research candidates. SgRNAs targeting the genome of each miRNA were designed and NB4 cells with inducible expression of Cas9 protein were generated. After transduced sgRNA into NB4/Cas9 cells, the mutation level by PCR and surveyor assay were evaluated. The cell differentiation level was investigated by surface CD11b expression via flow cytometry. RESULTS: A total of 410 mature miRNAs which expressed in NB4 cells were detected out after treated by ATRA, 74 miRNAs were up-regulated and 55 were down-regulated miRNAs with DNA cleavage generated by CRISPR/Cas9 was assayed directly by PCR or surveyor assay, quantitative PCR showed that the expression of miRNA was downregulated, which evaluated that gene edition successfully inhibitied the expression of mature miRNA. MiR-223 knockout showed the myeloid differentation of NB4 significantly inhibitied, while miRNA-155 knockout showed the myeloid differentation of NB4 cells significantly increased. CONCLUSION: CRISPR/Cas9 is a powerful tool for gene editing and can lead to miRNA knockout. Knockouts of miR-223 and miR-155 have shown a differentiation-related phenotype, and the potential mechanism is the integrative regulation of target genes.


Asunto(s)
Edición Génica , MicroARNs , Sistemas CRISPR-Cas , Diferenciación Celular , MicroARNs/genética , Análisis de Secuencia de ARN , Tretinoina
3.
Nat Med ; 27(3): 546-559, 2021 03.
Artículo en Inglés | MEDLINE | ID: mdl-33654293

RESUMEN

Angiotensin-converting enzyme 2 (ACE2) and accessory proteases (TMPRSS2 and CTSL) are needed for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) cellular entry, and their expression may shed light on viral tropism and impact across the body. We assessed the cell-type-specific expression of ACE2, TMPRSS2 and CTSL across 107 single-cell RNA-sequencing studies from different tissues. ACE2, TMPRSS2 and CTSL are coexpressed in specific subsets of respiratory epithelial cells in the nasal passages, airways and alveoli, and in cells from other organs associated with coronavirus disease 2019 (COVID-19) transmission or pathology. We performed a meta-analysis of 31 lung single-cell RNA-sequencing studies with 1,320,896 cells from 377 nasal, airway and lung parenchyma samples from 228 individuals. This revealed cell-type-specific associations of age, sex and smoking with expression levels of ACE2, TMPRSS2 and CTSL. Expression of entry factors increased with age and in males, including in airway secretory cells and alveolar type 2 cells. Expression programs shared by ACE2+TMPRSS2+ cells in nasal, lung and gut tissues included genes that may mediate viral entry, key immune functions and epithelial-macrophage cross-talk, such as genes involved in the interleukin-6, interleukin-1, tumor necrosis factor and complement pathways. Cell-type-specific expression patterns may contribute to the pathogenesis of COVID-19, and our work highlights putative molecular pathways for therapeutic intervention.


Asunto(s)
/epidemiología , Interacciones Huésped-Patógeno/genética , Análisis de Secuencia de ARN/estadística & datos numéricos , Análisis de la Célula Individual/estadística & datos numéricos , Internalización del Virus , Adulto , Anciano , Anciano de 80 o más Años , Células Epiteliales Alveolares/metabolismo , Células Epiteliales Alveolares/virología , /metabolismo , /virología , Catepsina L/genética , Catepsina L/metabolismo , Conjuntos de Datos como Asunto/estadística & datos numéricos , Demografía , Femenino , Perfilación de la Expresión Génica/estadística & datos numéricos , Humanos , Pulmón/metabolismo , Pulmón/virología , Masculino , Persona de Mediana Edad , Especificidad de Órganos/genética , Sistema Respiratorio/metabolismo , Sistema Respiratorio/virología , Análisis de Secuencia de ARN/métodos , Serina Endopeptidasas/genética , Serina Endopeptidasas/metabolismo , Análisis de la Célula Individual/métodos
4.
Nat Commun ; 12(1): 1588, 2021 03 11.
Artículo en Inglés | MEDLINE | ID: mdl-33707431

RESUMEN

Adipose tissue expansion, as seen in obesity, is often metabolically detrimental causing insulin resistance and the metabolic syndrome. However, white adipose tissue expansion at early ages is essential to establish a functional metabolism. To understand the differences between adolescent and adult adipose tissue expansion, we studied the cellular composition of the stromal vascular fraction of subcutaneous adipose tissue of two and eight weeks old mice using single cell RNA sequencing. We identified a subset of adolescent preadipocytes expressing the mature white adipocyte marker Asc-1 that showed a low ability to differentiate into beige adipocytes compared to Asc-1 negative cells in vitro. Loss of Asc-1 in subcutaneous preadipocytes resulted in spontaneous differentiation of beige adipocytes in vitro and in vivo. Mechanistically, this was mediated by a function of the amino acid transporter ASC-1 specifically in proliferating preadipocytes involving the intracellular accumulation of the ASC-1 cargo D-serine.


Asunto(s)
Adipocitos Beige/metabolismo , Adipocitos Blancos/metabolismo , Tejido Adiposo Beige/crecimiento & desarrollo , Tejido Adiposo Blanco/crecimiento & desarrollo , Sistema de Transporte de Aminoácidos y+/metabolismo , Adipocitos Beige/citología , Adipocitos Blancos/citología , Tejido Adiposo Beige/citología , Tejido Adiposo Blanco/citología , Sistema de Transporte de Aminoácidos y+/genética , Animales , Secuencia de Bases , Diferenciación Celular/genética , Células Cultivadas , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Noqueados , Análisis de Secuencia de ARN , Análisis de la Célula Individual , Proteína Desacopladora 1/biosíntesis
6.
Front Immunol ; 12: 625881, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-33717140

RESUMEN

T cells play a critical role in coronavirus diseases. How they do so in COVID-19 may be revealed by analyzing the epigenetic chromatin accessibility of cis- and trans-regulatory elements and creating transcriptomic immune profiles. We performed single-cell assay for transposase-accessible chromatin (scATAC) and single-cell RNA (scRNA) sequencing (seq) on the peripheral blood mononuclear cells (PBMCs) of severely ill/critical patients (SCPs) infected with COVID-19, moderate patients (MPs), and healthy volunteer controls (HCs). About 76,570 and 107,862 single cells were used, respectively, for analyzing the characteristics of chromatin accessibility and transcriptomic immune profiles by the application of scATAC-seq (nine cases) and scRNA-seq (15 cases). The scATAC-seq detected 28,535 different peaks in the three groups; among these peaks, 41.6 and 10.7% were located in the promoter and enhancer regions, respectively. Compared to HCs, among the peak-located genes in the total T cells and its subsets, CD4+ T and CD8+ T cells, from SCPs and MPs were enriched with inflammatory pathways, such as mitogen-activated protein kinase (MAPK) signaling pathway and tumor necrosis factor (TNF) signaling pathway. The motifs of TBX21 were less accessible in the CD4+ T cells of SCPs compared with those in MPs. Furthermore, the scRNA-seq showed that the proportion of T cells, especially the CD4+ T cells, was decreased in SCPs and MPs compared with those in HCs. Transcriptomic results revealed that histone-related genes, and inflammatory genes, such as NFKBIA, S100A9, and PIK3R1, were highly expressed in the total T cells, CD4+ T and CD8+ T cells, both in the cases of SCPs and MPs. In the CD4+ T cells, decreased T helper-1 (Th1) cells were observed in SCPs and MPs. In the CD8+T cells, activation markers, such as CD69 and HLA class II genes (HLA-DRA, HLA-DRB1, and HLA-DRB5), were significantly upregulated in SCPs. An integrated analysis of the data from scATAC-seq and scRNA-seq showed some consistency between the approaches. Cumulatively, we have generated a landscape of chromatin epigenetic status and transcriptomic immune profiles of T cells in patients with COVID-19. This has provided a deeper dissection of the characteristics of the T cells involved at a higher resolution than from previously obtained data merely by the scRNA-seq analysis. Our data led us to suggest that the T-cell inflammatory states accompanied with defective functions in the CD4+ T cells of SCPs may be the key factors for determining the pathogenesis of and recovery from COVID-19.


Asunto(s)
Linfocitos T CD4-Positivos/fisiología , Linfocitos T CD8-positivos/fisiología , Cromatina/metabolismo , /fisiología , /genética , Calgranulina B/genética , Cromatina/genética , Fosfatidilinositol 3-Quinasa Clase Ia/genética , Epigenoma/inmunología , Perfilación de la Expresión Génica , Humanos , Inmunidad Celular/genética , Inflamación/genética , Activación de Linfocitos , Inhibidor NF-kappaB alfa/genética , Análisis de Secuencia de ARN , Análisis de la Célula Individual , Transposasas/metabolismo , Regulación hacia Arriba
8.
Nat Commun ; 12(1): 1451, 2021 03 01.
Artículo en Inglés | MEDLINE | ID: mdl-33649316

RESUMEN

Proprioceptive feedback mainly derives from groups Ia and II muscle spindle (MS) afferents and group Ib Golgi tendon organ (GTO) afferents, but the molecular correlates of these three afferent subtypes remain unknown. We performed single cell RNA sequencing of genetically identified adult proprioceptors and uncovered five molecularly distinct neuronal clusters. Validation of cluster-specific transcripts in dorsal root ganglia and skeletal muscle demonstrates that two of these clusters correspond to group Ia MS afferents and group Ib GTO afferent proprioceptors, respectively, and suggest that the remaining clusters could represent group II MS afferents. Lineage analysis between proprioceptor transcriptomes at different developmental stages provides evidence that proprioceptor subtype identities emerge late in development. Together, our data provide comprehensive molecular signatures for groups Ia and II MS afferents and group Ib GTO afferents, enabling genetic interrogation of the role of individual proprioceptor subtypes in regulating motor output.


Asunto(s)
Mecanorreceptores/metabolismo , Husos Musculares/metabolismo , Neuronas Aferentes/metabolismo , Animales , Calbindina 2/metabolismo , Fenómenos Electrofisiológicos , Canales Iónicos/metabolismo , Ratones Transgénicos , Neuronas/metabolismo , Propiocepción , ARN Mensajero/genética , ARN Mensajero/metabolismo , Receptores de Neurotransmisores/metabolismo , Reproducibilidad de los Resultados , Análisis de Secuencia de ARN , Análisis de la Célula Individual , Transcriptoma/genética
9.
Nat Commun ; 12(1): 1565, 2021 03 10.
Artículo en Inglés | MEDLINE | ID: mdl-33692365

RESUMEN

During late lung development, alveolar and microvascular development is finalized to enable sufficient gas exchange. Impaired late lung development manifests as bronchopulmonary dysplasia (BPD) in preterm infants. Single-cell RNA sequencing (scRNA-seq) allows for assessment of complex cellular dynamics during biological processes, such as development. Here, we use MULTI-seq to generate scRNA-seq profiles of over 66,000 cells from 36 mice during normal or impaired lung development secondary to hyperoxia with validation of some of the findings in lungs from BPD patients. We observe dynamic populations of cells, including several rare cell types and putative progenitors. Hyperoxia exposure, which mimics the BPD phenotype, alters the composition of all cellular compartments, particularly alveolar epithelium, stromal fibroblasts, capillary endothelium and macrophage populations. Pathway analysis and predicted dynamic cellular crosstalk suggest inflammatory signaling as the main driver of hyperoxia-induced changes. Our data provides a single-cell view of cellular changes associated with late lung development in health and disease.


Asunto(s)
Hiperoxia/genética , Hiperoxia/fisiopatología , Pulmón/metabolismo , Pulmón/patología , Análisis de Secuencia de ARN/métodos , Análisis de la Célula Individual/métodos , Animales , Displasia Broncopulmonar/genética , Displasia Broncopulmonar/patología , Genotipo , Masculino , Ratones
10.
J Vis Exp ; (168)2021 02 23.
Artículo en Inglés | MEDLINE | ID: mdl-33720114

RESUMEN

Although small regulatory RNAs (sRNAs) are widespread among the bacterial domain of life, the functions of many of them remain poorly characterized notably due to the difficulty of identifying their mRNA targets. Here, we described a modified protocol of the MS2-Affinity Purification coupled with RNA Sequencing (MAPS) technology, aiming to reveal all RNA partners of a specific sRNA in vivo. Broadly, the MS2 aptamer is fused to the 5' extremity of the sRNA of interest. This construct is then expressed in vivo, allowing the MS2-sRNA to interact with its cellular partners. After bacterial harvesting, cells are mechanically lysed. The crude extract is loaded into an amylose-based chromatography column previously coated with the MS2 protein fused to the maltose binding protein. This enables the specific capture of MS2-sRNA and interacting RNAs. After elution, co-purified RNAs are identified by high-throughput RNA sequencing and subsequent bioinformatic analysis. The following protocol has been implemented in the Gram-positive human pathogen Staphylococcus aureus and is, in principle, transposable to any Gram-positive bacteria. To sum up, MAPS technology constitutes an efficient method to deeply explore the regulatory network of a particular sRNA, offering a snapshot of its whole targetome. However, it is important to keep in mind that putative targets identified by MAPS still need to be validated by complementary experimental approaches.


Asunto(s)
Aptámeros de Nucleótidos/metabolismo , Cromatografía de Afinidad , Bacterias Grampositivas/genética , Análisis de Secuencia de ARN , Secuencia de Bases , Tampones (Química) , Fraccionamiento Celular , Análisis de Datos , Regulación Bacteriana de la Expresión Génica , Humanos , Plásmidos/genética , ARN Bacteriano/genética , ARN Bacteriano/aislamiento & purificación , ARN Mensajero/genética , ARN Mensajero/metabolismo , ARN Pequeño no Traducido/genética , Reproducibilidad de los Resultados , Staphylococcus aureus/genética
11.
BMC Bioinformatics ; 22(1): 146, 2021 Mar 22.
Artículo en Inglés | MEDLINE | ID: mdl-33752598

RESUMEN

BACKGROUND: Polyploidy is very common in plants and can be seen as one of the key drivers in the domestication of crops and the establishment of important agronomic traits. It can be the main source of genomic repatterning and introduces gene duplications, affecting gene expression and alternative splicing. Since fully sequenced genomes are not yet available for many plant species including crops, de novo transcriptome assembly is the basis to understand molecular and functional mechanisms. However, in complex polyploid plants, de novo transcriptome assembly is challenging, leading to increased rates of fused or redundant transcripts. Since assemblers were developed mainly for diploid organisms, they may not well suited for polyploids. Also, comparative evaluations of these tools on higher polyploid plants are extremely rare. Thus, our aim was to fill this gap and to provide a basic guideline for choosing the optimal de novo assembly strategy focusing on autotetraploids, as the scientific interest in this type of polyploidy is steadily increasing. RESULTS: We present a comparison of two common (SOAPdenovo-Trans, Trinity) and one recently published transcriptome assembler (TransLiG) on diploid and autotetraploid species of the genera Acer and Vaccinium using Arabidopsis thaliana as a reference. The number of assembled transcripts was up to 11 and 14 times higher with an increased number of short transcripts for Acer and Vaccinium, respectively, compared to A. thaliana. In diploid samples, Trinity and TransLiG performed similarly good while in autotetraploids, TransLiG assembled most complete transcriptomes with an average of 1916 assembled BUSCOs vs. 1705 BUSCOs for Trinity. Of all three assemblers, SOAPdenovo-Trans performed worst (1133 complete BUSCOs). CONCLUSION: All three assembly tools produced complete assemblies when dealing with the model organism A. thaliana, independently of its ploidy level, but their performances differed extremely when it comes to non-model autotetraploids, where specifically TransLiG and Trinity produced a high number of redundant transcripts. The recently published assembler TransLiG has not been tested yet on any plant organism but showed highest completeness and full-length transcriptomes, especially in autotetraploids. Including such species during the development and testing of new assembly tools is highly appreciated and recommended as many important crops are polyploid.


Asunto(s)
Secuenciación de Nucleótidos de Alto Rendimiento , Transcriptoma , Perfilación de la Expresión Génica , Humanos , Poliploidía , Análisis de Secuencia de ARN
12.
Nat Commun ; 12(1): 1873, 2021 03 25.
Artículo en Inglés | MEDLINE | ID: mdl-33767149

RESUMEN

Clustering is a critical step in single cell-based studies. Most existing methods support unsupervised clustering without the a priori exploitation of any domain knowledge. When confronted by the high dimensionality and pervasive dropout events of scRNA-Seq data, purely unsupervised clustering methods may not produce biologically interpretable clusters, which complicates cell type assignment. In such cases, the only recourse is for the user to manually and repeatedly tweak clustering parameters until acceptable clusters are found. Consequently, the path to obtaining biologically meaningful clusters can be ad hoc and laborious. Here we report a principled clustering method named scDCC, that integrates domain knowledge into the clustering step. Experiments on various scRNA-seq datasets from thousands to tens of thousands of cells show that scDCC can significantly improve clustering performance, facilitating the interpretability of clusters and downstream analyses, such as cell type assignment.


Asunto(s)
Perfilación de la Expresión Génica/métodos , RNA-Seq/métodos , Análisis de Secuencia de ARN/métodos , Análisis de la Célula Individual/métodos , Animales , Caenorhabditis elegans/genética , Análisis por Conglomerados , Bases de Datos Genéticas , Humanos , Hígado/citología , Ratones , Transcriptoma/genética
13.
J Biomed Nanotechnol ; 17(2): 196-204, 2021 Feb 28.
Artículo en Inglés | MEDLINE | ID: mdl-33785091

RESUMEN

Heterotopic ossification is a bona fide bone formation outside the normal skeleton. Traumatic injury and genetic mutations are the important risk factors of HO. Both injury-induced HO and hereditary HO severely affect human life quality. However, there were no effect therapies treating HO. Here, we performed the RNA-sequencing assay to examine dynamic process during HO initiation and development. Moreover, we found that oxidation-reduction process were significantly dysregulated following HO formation. Further, we characterized that Nuclear factor erythroid 2-related factor 2 (NRF2) expression conferred antioxidant property in macrophages and then helped chondrocytes formation. Instead, 3-Nitrotyrosine is expressed by T-lymphocytes, but not macrophages, causing deficient adaptive immunity. Inhibition of NRF2 markedly alleviated HO. Finally, we identified that PI3K/AKT signaling pathway is responsible for whole HO process, but ERK signaling is activated only in early stage. ERK pathway blockade effectively prevented HO. These findings revealed that oxidative stress induced by early immune response can be targeted in HO treatment.


Asunto(s)
Osificación Heterotópica , Fosfatidilinositol 3-Quinasas , Hemo-Oxigenasa 1/genética , Hemo-Oxigenasa 1/metabolismo , Humanos , Osificación Heterotópica/genética , Osificación Heterotópica/prevención & control , Estrés Oxidativo , Fosfatidilinositol 3-Quinasas/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Análisis de Secuencia de ARN
14.
Nat Genet ; 53(3): 332-341, 2021 03.
Artículo en Inglés | MEDLINE | ID: mdl-33649592

RESUMEN

Resistance to immune checkpoint inhibitors (ICIs) is a key challenge in cancer therapy. To elucidate underlying mechanisms, we developed Perturb-CITE-sequencing (Perturb-CITE-seq), enabling pooled clustered regularly interspaced short palindromic repeat (CRISPR)-Cas9 perturbations with single-cell transcriptome and protein readouts. In patient-derived melanoma cells and autologous tumor-infiltrating lymphocyte (TIL) co-cultures, we profiled transcriptomes and 20 proteins in ~218,000 cells under ~750 perturbations associated with cancer cell-intrinsic ICI resistance (ICR). We recover known mechanisms of resistance, including defects in the interferon-γ (IFN-γ)-JAK/STAT and antigen-presentation pathways in RNA, protein and perturbation space, and new ones, including loss/downregulation of CD58. Loss of CD58 conferred immune evasion in multiple co-culture models and was downregulated in tumors of melanoma patients with ICR. CD58 protein expression was not induced by IFN-γ signaling, and CD58 loss conferred immune evasion without compromising major histocompatibility complex (MHC) expression, suggesting that it acts orthogonally to known mechanisms of ICR. This work provides a framework for the deciphering of complex mechanisms by large-scale perturbation screens with multimodal, single-cell readouts, and discovers potentially clinically relevant mechanisms of immune evasion.


Asunto(s)
Antígenos CD58/inmunología , Resistencia a Antineoplásicos/inmunología , Melanoma/patología , Análisis de la Célula Individual/métodos , Escape del Tumor , Antígenos CD58/genética , Antígenos CD58/metabolismo , Sistemas CRISPR-Cas , Técnicas de Cocultivo , Biología Computacional/métodos , Resistencia a Antineoplásicos/efectos de los fármacos , Resistencia a Antineoplásicos/genética , Epítopos/genética , Técnicas de Inactivación de Genes , Humanos , Interferón gamma/inmunología , Interferón gamma/metabolismo , Linfocitos Infiltrantes de Tumor/patología , Melanoma/tratamiento farmacológico , Melanoma/inmunología , Análisis de Secuencia de ARN , Escape del Tumor/genética
15.
Nat Genet ; 53(3): 304-312, 2021 03.
Artículo en Inglés | MEDLINE | ID: mdl-33664506

RESUMEN

Studying the function of common genetic variants in primary human tissues and during development is challenging. To address this, we use an efficient multiplexing strategy to differentiate 215 human induced pluripotent stem cell (iPSC) lines toward a midbrain neural fate, including dopaminergic neurons, and use single-cell RNA sequencing (scRNA-seq) to profile over 1 million cells across three differentiation time points. The proportion of neurons produced by each cell line is highly reproducible and is predictable by robust molecular markers expressed in pluripotent cells. Expression quantitative trait loci (eQTL) were characterized at different stages of neuronal development and in response to rotenone-induced oxidative stress. Of these, 1,284 eQTL colocalize with known neurological trait risk loci, and 46% are not found in the Genotype-Tissue Expression (GTEx) catalog. Our study illustrates how coupling scRNA-seq with long-term iPSC differentiation enables mechanistic studies of human trait-associated genetic variants in otherwise inaccessible cell states.


Asunto(s)
Neuronas Dopaminérgicas/citología , Neuronas Dopaminérgicas/fisiología , Células Madre Pluripotentes Inducidas/citología , Sitios de Carácter Cuantitativo , Transcriptoma , Diferenciación Celular/genética , Predisposición Genética a la Enfermedad , Humanos , Células Madre Pluripotentes Inducidas/fisiología , Neurogénesis/genética , Estrés Oxidativo/efectos de los fármacos , Receptor Tipo 1 de Factor de Crecimiento de Fibroblastos/genética , Rotenona/toxicidad , Análisis de Secuencia de ARN , Análisis de la Célula Individual
16.
Nat Genet ; 53(3): 313-321, 2021 03.
Artículo en Inglés | MEDLINE | ID: mdl-33664507

RESUMEN

Induced pluripotent stem cells (iPSCs) are an established cellular system to study the impact of genetic variants in derived cell types and developmental contexts. However, in their pluripotent state, the disease impact of genetic variants is less well known. Here, we integrate data from 1,367 human iPSC lines to comprehensively map common and rare regulatory variants in human pluripotent cells. Using this population-scale resource, we report hundreds of new colocalization events for human traits specific to iPSCs, and find increased power to identify rare regulatory variants compared with somatic tissues. Finally, we demonstrate how iPSCs enable the identification of causal genes for rare diseases.


Asunto(s)
Variación Genética , Células Madre Pluripotentes Inducidas/fisiología , Sitios de Carácter Cuantitativo , Síndrome de Bardet-Biedl/genética , Canales de Calcio/genética , Línea Celular , Ataxia Cerebelosa/genética , Metilación de ADN , Expresión Génica , Humanos , Células Madre Pluripotentes Inducidas/citología , Polimorfismo de Nucleótido Simple , Proteínas/genética , Enfermedades Raras/genética , Secuencias Reguladoras de Ácidos Nucleicos , Análisis de Secuencia de ARN , Secuenciación Completa del Genoma
17.
Nat Commun ; 12(1): 1652, 2021 03 12.
Artículo en Inglés | MEDLINE | ID: mdl-33712618

RESUMEN

Annotation of polyadenylation sites from short-read RNA sequencing alone is a challenging computational task. Other algorithms rooted in DNA sequence predict potential polyadenylation sites; however, in vivo expression of a particular site varies based on a myriad of conditions. Here, we introduce aptardi (alternative polyadenylation transcriptome analysis from RNA-Seq data and DNA sequence information), which leverages both DNA sequence and RNA sequencing in a machine learning paradigm to predict expressed polyadenylation sites. Specifically, as input aptardi takes DNA nucleotide sequence, genome-aligned RNA-Seq data, and an initial transcriptome. The program evaluates these initial transcripts to identify expressed polyadenylation sites in the biological sample and refines transcript 3'-ends accordingly. The average precision of the aptardi model is twice that of a standard transcriptome assembler. In particular, the recall of the aptardi model (the proportion of true polyadenylation sites detected by the algorithm) is improved by over three-fold. Also, the model-trained using the Human Brain Reference RNA commercial standard-performs well when applied to RNA-sequencing samples from different tissues and different mammalian species. Finally, aptardi's input is simple to compile and its output is easily amenable to downstream analyses such as quantitation and differential expression.


Asunto(s)
Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Poliadenilación , Transcriptoma , Animales , Secuencia de Bases , Perfilación de la Expresión Génica , Humanos , ARN/química , ARN/metabolismo , Análisis de Secuencia de ARN , Biología de Sistemas
18.
Nat Commun ; 12(1): 1771, 2021 03 19.
Artículo en Inglés | MEDLINE | ID: mdl-33741943

RESUMEN

Single-cell RNA sequencing is a powerful tool to study developmental biology but does not preserve spatial information about tissue morphology and cellular interactions. Here, we combine single-cell and spatial transcriptomics with algorithms for data integration to study the development of the chicken heart from the early to late four-chambered heart stage. We create a census of the diverse cellular lineages in developing hearts, their spatial organization, and their interactions during development. Spatial mapping of differentiation transitions in cardiac lineages defines transcriptional differences between epithelial and mesenchymal cells within the epicardial lineage. Using spatially resolved expression analysis, we identify anatomically restricted expression programs, including expression of genes implicated in congenital heart disease. Last, we discover a persistent enrichment of the small, secreted peptide, thymosin beta-4, throughout coronary vascular development. Overall, our study identifies an intricate interplay between cellular differentiation and morphogenesis.


Asunto(s)
Regulación del Desarrollo de la Expresión Génica , Corazón/embriología , Morfogénesis/genética , Miocardio/metabolismo , Análisis de Secuencia de ARN/métodos , Análisis de la Célula Individual/métodos , Animales , Diferenciación Celular/genética , Linaje de la Célula/genética , Embrión de Pollo , Pollos , Células Epiteliales/metabolismo , Perfilación de la Expresión Génica/métodos , Humanos , Mesodermo/citología , Mesodermo/embriología , Mesodermo/metabolismo , Miocardio/citología
19.
Nat Commun ; 12(1): 1758, 2021 03 19.
Artículo en Inglés | MEDLINE | ID: mdl-33741948

RESUMEN

The molecular machinery and chromosome structures carrying out meiosis are frequently conserved from yeast to mammals. However, signals initiating meiosis appear divergent: while nutrient restriction induces meiosis in the yeast system, retinoic acid (RA) and its target Stra8 have been shown to be necessary but not sufficient to induce meiotic initiation in mammalian germ cells. Here, we use primary culture of mouse undifferentiated spermatogonia without the support of gonadal somatic cells to show that nutrient restriction in combination with RA is sufficient to induce Stra8- and Spo11-dependent meiotic gene and chromosome programs that recapitulate the transcriptomic and cytologic features of in vivo meiosis. We demonstrate that neither nutrient restriction nor RA alone exerts these effects. Moreover, we identify a distinctive network of 11 nutrient restriction-upregulated transcription factor genes, which are associated with early meiosis in vivo and whose expression does not require RA. Our study proposes a conserved model, in which nutrient restriction induces meiotic initiation by upregulating key transcription factor genes for the meiotic gene program and provides an in vitro platform for meiotic induction that could facilitate research and haploid gamete production.


Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Endodesoxirribonucleasas/metabolismo , Meiosis/efectos de los fármacos , Nutrientes/metabolismo , Espermatogonias/efectos de los fármacos , Tretinoina/farmacología , Proteínas Adaptadoras Transductoras de Señales/genética , Animales , Células Cultivadas , Endodesoxirribonucleasas/genética , Expresión Génica/efectos de los fármacos , Masculino , Mamíferos/genética , Mamíferos/metabolismo , Meiosis/genética , Ratones Endogámicos C57BL , Ratones Endogámicos DBA , Ratones Noqueados , Análisis de Secuencia de ARN/métodos , Análisis de la Célula Individual/métodos , Espermatogonias/citología , Espermatogonias/metabolismo
20.
Nat Commun ; 12(1): 1540, 2021 03 09.
Artículo en Inglés | MEDLINE | ID: mdl-33750785

RESUMEN

The tumor microenvironment (TME) of nasopharyngeal carcinoma (NPC) harbors a heterogeneous and dynamic stromal population. A comprehensive understanding of this tumor-specific ecosystem is necessary to enhance cancer diagnosis, therapeutics, and prognosis. However, recent advances based on bulk RNA sequencing remain insufficient to construct an in-depth landscape of infiltrating stromal cells in NPC. Here we apply single-cell RNA sequencing to 66,627 cells from 14 patients, integrated with clonotype identification on T and B cells. We identify and characterize five major stromal clusters and 36 distinct subpopulations based on genetic profiling. By comparing with the infiltrating cells in the non-malignant microenvironment, we report highly representative features in the TME, including phenotypic abundance, genetic alternations, immune dynamics, clonal expansion, developmental trajectory, and molecular interactions that profoundly influence patient prognosis and therapeutic outcome. The key findings are further independently validated in two single-cell RNA sequencing cohorts and two bulk RNA-sequencing cohorts. In the present study, we reveal the correlation between NPC-specific characteristics and progression-free survival. Together, these data facilitate the understanding of the stromal landscape and immune dynamics in NPC patients and provides deeper insights into the development of prognostic biomarkers and therapeutic targets in the TME.


Asunto(s)
Carcinoma Nasofaríngeo/metabolismo , Neoplasias Nasofaríngeas/metabolismo , Microambiente Tumoral/fisiología , Linfocitos B , Fibroblastos , Regulación Neoplásica de la Expresión Génica , Humanos , Células Mieloides , Carcinoma Nasofaríngeo/genética , Carcinoma Nasofaríngeo/inmunología , Neoplasias Nasofaríngeas/genética , Neoplasias Nasofaríngeas/inmunología , Fenotipo , Pronóstico , Supervivencia sin Progresión , Análisis de Secuencia de ARN , Células del Estroma , Linfocitos T , Microambiente Tumoral/inmunología
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