RESUMEN
Proper transport of RNAs to synapses is essential for localized translation of proteins in response to synaptic signals and synaptic plasticity. Alzheimer's disease (AD) is a neurodegenerative disease characterized by accumulation of amyloid aggregates and hyperphosphorylated tau neurofibrillary tangles followed by widespread synapse loss. To understand whether RNA synaptic localization is impacted in AD, we performed RNA sequencing on synaptosomes and brain homogenates from AD patients and cognitively healthy controls. This resulted in the discovery of hundreds of mislocalized mRNAs in AD among frontal and temporal brain regions. Similar observations were found in an APPswe/PSEN1dE9 mouse model. Furthermore, major differences were observed among circular RNAs (circRNAs) localized to synapses in AD including two overlapping isoforms of circGSK3ß, one upregulated, and one downregulated. Expression of these distinct isoforms affected tau phosphorylation in neuronal cells substantiating the importance of circRNAs in the brain and pointing to a new class of therapeutic targets.
Asunto(s)
Enfermedad de Alzheimer , ARN Circular , ARN Mensajero , Sinapsis , Proteínas tau , Enfermedad de Alzheimer/genética , Enfermedad de Alzheimer/metabolismo , ARN Circular/genética , ARN Circular/metabolismo , Humanos , Animales , Sinapsis/metabolismo , Sinapsis/genética , ARN Mensajero/genética , ARN Mensajero/metabolismo , Ratones , Proteínas tau/metabolismo , Proteínas tau/genética , Fosforilación , Modelos Animales de Enfermedad , Encéfalo/metabolismo , Encéfalo/patología , Masculino , Neuronas/metabolismo , Ratones Transgénicos , Sinaptosomas/metabolismo , Femenino , AncianoRESUMEN
Understanding how genetic variants impact molecular phenotypes is a key goal of functional genomics, currently hindered by reliance on a single haploid reference genome. Here, we present the EN-TEx resource of 1,635 open-access datasets from four donors (â¼30 tissues × â¼15 assays). The datasets are mapped to matched, diploid genomes with long-read phasing and structural variants, instantiating a catalog of >1 million allele-specific loci. These loci exhibit coordinated activity along haplotypes and are less conserved than corresponding, non-allele-specific ones. Surprisingly, a deep-learning transformer model can predict the allele-specific activity based only on local nucleotide-sequence context, highlighting the importance of transcription-factor-binding motifs particularly sensitive to variants. Furthermore, combining EN-TEx with existing genome annotations reveals strong associations between allele-specific and GWAS loci. It also enables models for transferring known eQTLs to difficult-to-profile tissues (e.g., from skin to heart). Overall, EN-TEx provides rich data and generalizable models for more accurate personal functional genomics.
Asunto(s)
Epigenoma , Sitios de Carácter Cuantitativo , Estudio de Asociación del Genoma Completo , Genómica , Fenotipo , Polimorfismo de Nucleótido SimpleRESUMEN
Short-read sequencers provide highly accurate reads at very low cost. Unfortunately, short reads are often inadequate for important applications such as assembly in complex regions or phasing across distant heterozygous sites. In this study, we describe novel bench protocols and algorithms to obtain haplotype-phased sequence assemblies with ultra-low error for regions 10 kb and longer using short reads only. We accomplish this by imprinting each template strand from a target region with a dense and unique mutation pattern. The mutation process randomly and independently converts â¼50% of cytosines to uracils. Sequencing libraries are made from both mutated and unmutated templates. Using de Bruijn graphs and paired-end read information, we assemble each mutated template and use the unmutated library to correct the mutated bases. Templates are partitioned into two or more haplotypes, and the final haplotypes are assembled and corrected for residual template mutations and PCR errors. With sufficient template coverage, the final assemblies have per-base error rates below 10-9. We demonstrate this method on a four-member nuclear family, correctly assembling and phasing three genomic intervals, including the highly polymorphic HLA-B gene.
Asunto(s)
Genoma , Genómica , Algoritmos , Antígenos HLA-B , Haplotipos , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Mutagénesis , Análisis de Secuencia de ADN/métodosRESUMEN
We have produced RNA sequencing data for 53 primary cells from different locations in the human body. The clustering of these primary cells reveals that most cells in the human body share a few broad transcriptional programs, which define five major cell types: epithelial, endothelial, mesenchymal, neural, and blood cells. These act as basic components of many tissues and organs. Based on gene expression, these cell types redefine the basic histological types by which tissues have been traditionally classified. We identified genes whose expression is specific to these cell types, and from these genes, we estimated the contribution of the major cell types to the composition of human tissues. We found this cellular composition to be a characteristic signature of tissues and to reflect tissue morphological heterogeneity and histology. We identified changes in cellular composition in different tissues associated with age and sex, and found that departures from the normal cellular composition correlate with histological phenotypes associated with disease.
Asunto(s)
Transcripción Genética , Línea Celular , Células Endoteliales/metabolismo , Células Epiteliales/metabolismo , Femenino , Perfilación de la Expresión Génica , Ginecomastia/genética , Ginecomastia/metabolismo , Humanos , Masculino , Mesodermo/citología , Mesodermo/metabolismo , Neoplasias/genética , Especificidad de Órganos , Análisis de Secuencia de ARNRESUMEN
We show the use of 5'-Acrydite oligonucleotides to copolymerize single-cell DNA or RNA into balls of acrylamide gel (BAGs). Combining this step with split-and-pool techniques for creating barcodes yields a method with advantages in cost and scalability, depth of coverage, ease of operation, minimal cross-contamination, and efficient use of samples. We perform DNA copy number profiling on mixtures of cell lines, nuclei from frozen prostate tumors, and biopsy washes. As applied to RNA, the method has high capture efficiency of transcripts and sufficient consistency to clearly distinguish the expression patterns of cell lines and individual nuclei from neurons dissected from the mouse brain. By using varietal tags (UMIs) to achieve sequence error correction, we show extremely low levels of cross-contamination by tracking source-specific SNVs. The method is readily modifiable, and we will discuss its adaptability and diverse applications.