RESUMO
BACKGROUND: Recently, measurement of RNA at single cell resolution has yielded surprising insights. Methods for single-cell RNA sequencing (scRNA-seq) have received considerable attention, but the broad reliability of single cell methods and the factors governing their performance are still poorly known. RESULTS: Here, we conducted a large-scale control experiment to assess the transfer function of three scRNA-seq methods and factors modulating the function. All three methods detected greater than 70% of the expected number of genes and had a 50% probability of detecting genes with abundance greater than 2 to 4 molecules. Despite the small number of molecules, sequencing depth significantly affected gene detection. While biases in detection and quantification were qualitatively similar across methods, the degree of bias differed, consistent with differences in molecular protocol. Measurement reliability increased with expression level for all methods and we conservatively estimate measurements to be quantitative at an expression level greater than ~5-10 molecules. CONCLUSIONS: Based on these extensive control studies, we propose that RNA-seq of single cells has come of age, yielding quantitative biological information.
Assuntos
Sequenciamento de Nucleotídeos em Larga Escala , Técnicas de Amplificação de Ácido Nucleico , RNA/genética , Análise de Célula Única , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Análise de Sequência de RNA , Análise de Célula Única/métodosRESUMO
Rett syndrome is an X-linked neurodevelopmental disorder caused by mutation in the methyl-CpG-binding protein 2 gene (MECP2) in the majority of cases. We describe an RNA sequencing dataset of postmortem brain tissue samples from four females clinically diagnosed with Rett syndrome and four age-matched female donors. The dataset contains 16 transcriptomes, including two brain regions, temporal and cingulate cortex, for each individual. We compared our dataset with published transcriptomic analyses of postmortem brain tissue from Rett syndrome and found consistent gene expression alterations among regions of the cerebral cortex. Our data provide a valuable resource to explore the biology of the human brain in Rett syndrome.
Assuntos
Giro do Cíngulo/metabolismo , Síndrome de Rett/metabolismo , Lobo Temporal/metabolismo , Transcriptoma , Cadáver , Feminino , Humanos , Análise de Sequência de RNARESUMO
BACKGROUND: Genome-wide association studies of schizophrenia have demonstrated that variations in noncoding regions are responsible for most of the common variation heritability of the disease. It is hypothesized that these risk variants alter gene expression. Therefore, studying alterations in gene expression in schizophrenia may provide a direct approach to understanding the etiology of the disease. In this study we use cultured neural progenitor cells derived from olfactory neuroepithelium (CNON cells) as a genetically unaltered cellular model to elucidate the neurodevelopmental aspects of schizophrenia. METHODS: We performed a gene expression study using RNA sequencing of CNON cells from 111 control subjects and 144 individuals with schizophrenia. Differentially expressed genes were identified with DESeq2 software, using covariates to correct for sex, age, library batches, and 1 surrogate variable component. RESULTS: A total of 80 genes were differentially expressed (false discovery rate < 10%), showing enrichment in cell migration, cell adhesion, developmental process, synapse assembly, cell proliferation, and related Gene Ontology categories. Cadherin and Wnt signaling pathways were positive in overrepresentation test, and, in addition, many genes were specifically involved in WNT5A signaling. The differentially expressed genes were modestly, but significantly, enriched in the genes overlapping single nucleotide polymorphisms with genome-wide significant association from the Psychiatric Genomics Consortium genome-wide association study of schizophrenia. We also found substantial overlap with genes associated with other psychiatric disorders or brain development, enrichment in the same Gene Ontology categories as genes with mutations de novo in schizophrenia, and studies of induced pluripotent stem cell-derived neural progenitor cells. CONCLUSIONS: CNON cells are a good model of the neurodevelopmental aspects of schizophrenia and can be used to elucidate the etiology of the disorder.
Assuntos
Células-Tronco Pluripotentes Induzidas , Células-Tronco Neurais , Esquizofrenia , Expressão Gênica , Estudo de Associação Genômica Ampla , Humanos , Esquizofrenia/genética , Proteína Wnt-5aRESUMO
Hepatitis B viruses (HBVs), which are enveloped viruses with reverse-transcribed DNA genomes, constitute the family Hepadnaviridae. An outstanding feature of HBVs is their streamlined genome organization with extensive gene overlap. Remarkably, the â¼1,100 bp open reading frame (ORF) encoding the envelope proteins is fully nested within the ORF of the viral replicase P. Here, we report the discovery of a diversified family of fish viruses, designated nackednaviruses, which lack the envelope protein gene, but otherwise exhibit key characteristics of HBVs including genome replication via protein-primed reverse-transcription and utilization of structurally related capsids. Phylogenetic reconstruction indicates that these two virus families separated more than 400 million years ago before the rise of tetrapods. We show that HBVs are of ancient origin, descending from non-enveloped progenitors in fishes. Their envelope protein gene emerged de novo, leading to a major transition in viral lifestyle, followed by co-evolution with their hosts over geologic eras.
Assuntos
Evolução Molecular , Doenças dos Peixes/virologia , Hepadnaviridae/genética , Vírus da Hepatite B/genética , Hepatite B/virologia , Sequência de Aminoácidos , Animais , Capsídeo/química , Capsídeo/metabolismo , Peixes , Genoma Viral , Hepadnaviridae/química , Hepadnaviridae/classificação , Hepadnaviridae/isolamento & purificação , Vírus da Hepatite B/química , Vírus da Hepatite B/classificação , Vírus da Hepatite B/isolamento & purificação , Humanos , Dados de Sequência Molecular , Filogenia , Alinhamento de Sequência , Proteínas do Envelope Viral/química , Proteínas do Envelope Viral/genéticaRESUMO
RE1-Silencing Transcription factor (REST) has a well-established role in regulating transcription of genes important for neuronal development. Its role in cancer, though significant, is less well understood. We show that REST downregulation in weakly invasive MCF-7 breast cancer cells converts them to a more invasive phenotype, while REST overexpression in highly invasive MDA-MB-231 cells suppresses invasiveness. Surprisingly, the mechanism responsible for these phenotypic changes does not depend directly on the transcriptional function of REST protein. Instead, it is driven by previously unstudied mid-size (30-200 nt) non-coding RNAs (ncRNAs) derived from the first exon of an alternatively spliced REST transcript: REST-003. We show that processing of REST-003 into ncRNAs is controlled by an uncharacterized serine/arginine repeat-related protein, SRRM3. SRRM3 expression may be under REST-mediated transcriptional control, as it increases following REST downregulation. The SRRM3-dependent regulation of REST-003 processing into ncRNAs has many similarities to recently described promoter-associated small RNA-like processes. Targeting ncRNAs that control invasiveness could lead to new therapeutic approaches to limit breast cancer metastasis.
Assuntos
Neoplasias da Mama/genética , Invasividade Neoplásica/genética , Proteínas/genética , RNA não Traduzido/genética , Proteínas Repressoras/genética , Processamento Alternativo/genética , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Regulação para Baixo , Feminino , Humanos , Células MCF-7 , Invasividade Neoplásica/patologia , Metástase Neoplásica/genética , Interferência de RNA , RNA Interferente Pequeno/genéticaRESUMO
BACKGROUND: We examined the performance of three RNA-Sequencing library preparation protocols as a function of RNA integrity, comparing gene expressions between heat-degraded samples to their high-quality counterparts. This work is invaluable given the difficulty of obtaining high-quality RNA from tissues, particularly those from individuals with disease phenotypes. RESULTS: With the integrity of total RNA being a critical parameter for RNA-Sequencing analysis, degraded RNA can heavily influence the results of gene expression profiles. We discovered that gene expression read results are influenced by RNA quality when a common library construction protocol is used. These results are based on one technical experiment from a pool of 4 neural progenitor cell lines. CONCLUSIONS: The use of alternative protocols can allow samples with a wider range of RNA qualities to be used, facilitating the investigation of disease tissues.