RESUMEN
How the splicing machinery defines exons or introns as the spliced unit has remained a puzzle for 30 years. Here, we demonstrate that peripheral and central regions of the nucleus harbor genes with two distinct exon-intron GC content architectures that differ in the splicing outcome. Genes with low GC content exons, flanked by long introns with lower GC content, are localized in the periphery, and the exons are defined as the spliced unit. Alternative splicing of these genes results in exon skipping. In contrast, the nuclear center contains genes with a high GC content in the exons and short flanking introns. Most splicing of these genes occurs via intron definition, and aberrant splicing leads to intron retention. We demonstrate that the nuclear periphery and center generate different environments for the regulation of alternative splicing and that two sets of splicing factors form discrete regulatory subnetworks for the two gene architectures. Our study connects 3D genome organization and splicing, thus demonstrating that exon and intron definition modes of splicing occur in different nuclear regions.
Asunto(s)
Empalme Alternativo , Empalme del ARN , Composición de Base , Exones/genética , Intrones/genéticaRESUMEN
Alternative splicing (AS) contributes to proteome diversity. As splicing occurs cotranscriptionally, epigenetic determinants such as DNA methylation likely play a part in regulation of AS. Previously, we have shown that DNA methylation marks exons and that a loss of DNA methylation alters splicing patterns in a genome-wide manner. To investigate the influence of DNA methylation on splicing of individual genes, we developed a method to manipulate DNA methylation in vivo in a site-specific manner using the deactivated endonuclease Cas9 fused to enzymes that methylate or demethylate DNA. We used this system to directly change the DNA methylation pattern of selected exons and introns. We demonstrated that changes in the methylation pattern of alternatively spliced exons, but not constitutively spliced exons or introns, altered inclusion levels. This is the first direct demonstration that DNA methylation of exon-encoding regions is directly involved in regulation of AS.
Asunto(s)
Empalme Alternativo , Metilación de ADN , Exones , Sistemas CRISPR-Cas , Línea Celular , ADN (Citosina-5-)-Metiltransferasas/genética , ADN (Citosina-5-)-Metiltransferasas/metabolismo , ADN Metiltransferasa 3A , Edición Génica , Marcación de Gen , Humanos , Intrones , ARN Guía de KinetoplastidaRESUMEN
BACKGROUND: Olfaction is a versatile sensory mechanism for detecting thousands of volatile odorants. Although molecular basis of odorant signaling is relatively well understood considerable gaps remain in the complete charting of all relevant gene products. To address this challenge, we applied RNAseq to four well-characterized human olfactory epithelial samples and compared the results to novel and published mouse olfactory epithelium as well as 16 human control tissues. RESULTS: We identified 194 non-olfactory receptor (OR) genes that are overexpressed in human olfactory tissues vs. CONTROLS: The highest overexpression is seen for lipocalins and bactericidal/permeability-increasing (BPI)-fold proteins, which in other species include secreted odorant carriers. Mouse-human discordance in orthologous lipocalin expression suggests different mammalian evolutionary paths in this family. Of the overexpressed genes 36 have documented olfactory function while for 158 there is little or no previous such functional evidence. The latter group includes GPCRs, neuropeptides, solute carriers, transcription factors and biotransformation enzymes. Many of them may be indirectly implicated in sensory function, and ~70 % are over expressed also in mouse olfactory epithelium, corroborating their olfactory role. Nearly 90 % of the intact OR repertoire, and ~60 % of the OR pseudogenes are expressed in the olfactory epithelium, with the latter showing a 3-fold lower expression. ORs transcription levels show a 1000-fold inter-paralog variation, as well as significant inter-individual differences. We assembled 160 transcripts representing 100 intact OR genes. These include 1-4 short 5' non-coding exons with considerable alternative splicing and long last exons that contain the coding region and 3' untranslated region of highly variable length. Notably, we identified 10 ORs with an intact open reading frame but with seemingly non-functional transcripts, suggesting a yet unreported OR pseudogenization mechanism. Analysis of the OR upstream regions indicated an enrichment of the homeobox family transcription factor binding sites and a consensus localization of a specific transcription factor binding site subfamily (Olf/EBF). CONCLUSIONS: We provide an overview of expression levels of ORs and auxiliary genes in human olfactory epithelium. This forms a transcriptomic view of the entire OR repertoire, and reveals a large number of over-expressed uncharacterized human non-receptor genes, providing a platform for future discovery.
Asunto(s)
Lipocalinas/genética , Mucosa Olfatoria/metabolismo , ARN Mensajero/genética , Receptores Odorantes/genética , Olfato/genética , Transcriptoma , Animales , Autoantígenos/genética , Autoantígenos/metabolismo , Proteínas de Unión a Ácidos Grasos , Perfilación de la Expresión Génica , Regulación de la Expresión Génica , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Lipocalinas/clasificación , Lipocalinas/metabolismo , Proteínas de Transporte de Membrana/genética , Proteínas de Transporte de Membrana/metabolismo , Ratones , Neuropéptidos/genética , Neuropéptidos/metabolismo , Filogenia , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Proteínas/genética , Proteínas/metabolismo , Seudogenes , ARN Mensajero/metabolismo , Receptores Odorantes/metabolismo , Transducción de Señal , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
Genetic variations in olfactory receptors likely contribute to the diversity of odorant-specific sensitivity phenotypes. Our working hypothesis is that genetic variations in auxiliary olfactory genes, including those mediating transduction and sensory neuronal development, may constitute the genetic basis for general olfactory sensitivity (GOS) and congenital general anosmia (CGA). We thus performed a systematic exploration for auxiliary olfactory genes and their documented variation. This included a literature survey, seeking relevant functional in vitro studies, mouse gene knockouts and human disorders with olfactory phenotypes, as well as data mining in published transcriptome and proteome data for genes expressed in olfactory tissues. In addition, we performed next-generation transcriptome sequencing (RNA-seq) of human olfactory epithelium and mouse olfactory epithelium and bulb, so as to identify sensory-enriched transcripts. Employing a global score system based on attributes of the 11 data sources utilized, we identified a list of 1,680 candidate auxiliary olfactory genes, of which 450 are shortlisted as having higher probability of a functional role. For the top-scoring 136 genes, we identified genomic variants (probably damaging single nucleotide polymorphisms, indels, and copy number deletions) gleaned from public variation repositories. This database of genes and their variants should assist in rationalizing the great interindividual variation in human overall olfactory sensitivity (http://genome.weizmann.ac.il/GOSdb).
Asunto(s)
Bases de Datos Genéticas , Variación Genética , Genoma/genética , Receptores Odorantes/genética , Animales , Variaciones en el Número de Copia de ADN , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Mutación INDEL , Internet , Ratones , Bulbo Olfatorio/metabolismo , Mucosa Olfatoria/metabolismo , Polimorfismo de Nucleótido Simple , Proteoma/genética , Especificidad de la Especie , Transcriptoma/genéticaRESUMEN
Copy-number variations (CNVs) are widespread in the human genome, but comprehensive assignments of integer locus copy-numbers (i.e., copy-number genotypes) that, for example, enable discrimination of homozygous from heterozygous CNVs, have remained challenging. Here we present CopySeq, a novel computational approach with an underlying statistical framework that analyzes the depth-of-coverage of high-throughput DNA sequencing reads, and can incorporate paired-end and breakpoint junction analysis based CNV-analysis approaches, to infer locus copy-number genotypes. We benchmarked CopySeq by genotyping 500 chromosome 1 CNV regions in 150 personal genomes sequenced at low-coverage. The assessed copy-number genotypes were highly concordant with our performed qPCR experiments (Pearson correlation coefficient 0.94), and with the published results of two microarray platforms (95-99% concordance). We further demonstrated the utility of CopySeq for analyzing gene regions enriched for segmental duplications by comprehensively inferring copy-number genotypes in the CNV-enriched >800 olfactory receptor (OR) human gene and pseudogene loci. CopySeq revealed that OR loci display an extensive range of locus copy-numbers across individuals, with zero to two copies in some OR loci, and two to nine copies in others. Among genetic variants affecting OR loci we identified deleterious variants including CNVs and SNPs affecting ~15% and ~20% of the human OR gene repertoire, respectively, implying that genetic variants with a possible impact on smell perception are widespread. Finally, we found that for several OR loci the reference genome appears to represent a minor-frequency variant, implying a necessary revision of the OR repertoire for future functional studies. CopySeq can ascertain genomic structural variation in specific gene families as well as at a genome-wide scale, where it may enable the quantitative evaluation of CNVs in genome-wide association studies involving high-throughput sequencing.
Asunto(s)
Variaciones en el Número de Copia de ADN , Genómica/métodos , Receptores Odorantes/genética , Mapeo Cromosómico , Bases de Datos Genéticas , Variación Genética , Genética de Población , Genoma , Genotipo , Humanos , Análisis de Secuencia por Matrices de Oligonucleótidos , Reacción en Cadena de la Polimerasa , Polimorfismo de Nucleótido Simple , Análisis de Componente Principal , Reproducibilidad de los Resultados , Análisis de Secuencia de ADN , Programas Informáticos , Población BlancaRESUMEN
In the earliest step of spliceosome assembly, the two splice sites flanking an intron are brought into proximity by U1 snRNP and U2AF along with other proteins. The mechanism that facilitates this intron looping is poorly understood. Using a CRISPR interference-based approach to halt RNA polymerase II transcription in the middle of introns in human cells, we discovered that the nascent 5' splice site base pairs with a U1 snRNA that is tethered to RNA polymerase II during intron synthesis. This association functionally corresponds with splicing outcome, involves bona fide 5' splice sites and cryptic intronic sites, and occurs transcriptome-wide. Overall, our findings reveal that the upstream 5' splice sites remain attached to the transcriptional machinery during intron synthesis and are thus brought into proximity of the 3' splice sites; potentially mediating the rapid splicing of long introns.
Asunto(s)
Intrones/genética , Sitios de Empalme de ARN/genética , Transcripción Genética , Emparejamiento Base/genética , Secuencia de Bases , Exones/genética , Células HEK293 , Células HeLa , Humanos , Proteínas de Microfilamentos/genética , ARN Polimerasa II/metabolismo , Precursores del ARN/genética , Precursores del ARN/metabolismo , ARN Nuclear Pequeño/genética , ARN Nuclear Pequeño/metabolismo , Proteínas de Unión al ARN/genética , Ribonucleoproteína Nuclear Pequeña U1/metabolismo , Transcriptoma/genéticaRESUMEN
How gene expression correlates with schizophrenia across individuals is beginning to be examined through analyses of RNA-seq from postmortem brains of individuals with disease and control brains. Here we focus on variation in allele-specific expression, following up on the CommonMind Consortium (CMC) RNA-seq experiments of nearly 600 human dorsolateral prefrontal cortex (DLPFC) samples. Analyzing the extent of allelic expression bias-a hallmark of imprinting-we find that the number of imprinted human genes is consistent with lower estimates (≈0.5% of all genes), and thus contradicts much higher estimates. Moreover, the handful of putatively imprinted genes are all in close genomic proximity to known imprinted genes. Joint analysis of the imprinted genes across hundreds of individuals allowed us to establish how allelic bias depends on various factors. We find that age and genetic ancestry have gene-specific, differential effect on allelic bias. In contrast, allelic bias appears to be independent of schizophrenia.