RESUMEN
Pre-mRNA splicing is a fundamental step in gene expression, conserved across eukaryotes, in which the spliceosome recognizes motifs at the 3' and 5' splice sites (SSs), excises introns, and ligates exons. SS recognition and pairing is often influenced by protein splicing factors (SFs) that bind to splicing regulatory elements (SREs). Here, we describe SMsplice, a fully interpretable model of pre-mRNA splicing that combines models of core SS motifs, SREs, and exonic and intronic length preferences. We learn models that predict SS locations with 83 to 86% accuracy in fish, insects, and plants and about 70% in mammals. Learned SRE motifs include both known SF binding motifs and unfamiliar motifs, and both motif classes are supported by genetic analyses. Our comparisons across species highlight similarities between non-mammals, increased reliance on intronic SREs in plant splicing, and a greater reliance on SREs in mammalian splicing.
Asunto(s)
Exones , Intrones , Precursores del ARN , Sitios de Empalme de ARN , Empalme del ARN , Precursores del ARN/genética , Precursores del ARN/metabolismo , Animales , Intrones/genética , Exones/genética , Genes de Plantas , Modelos Genéticos , Empalmosomas/metabolismo , Empalmosomas/genética , Plantas/genética , Humanos , Factores de Empalme de ARN/genética , Factores de Empalme de ARN/metabolismoRESUMEN
Pathogenic variants in SCN8A, which encodes the voltage-gated sodium (NaV) channel NaV1.6, associate with neurodevelopmental disorders, including developmental and epileptic encephalopathy. Previous approaches to determine SCN8A variant function may be confounded by use of a neonatally expressed, alternatively spliced isoform of NaV1.6 (NaV1.6N) and engineered mutations rendering the channel tetrodotoxin (TTX) resistant. We investigated the impact of SCN8A alternative splicing on variant function by comparing the functional attributes of 15 variants expressed in 2 developmentally regulated splice isoforms (NaV1.6N, NaV1.6A). We employed automated patch clamp recording to enhance throughput, and developed a neuronal cell line (ND7/LoNav) with low levels of endogenous NaV current to obviate the need for TTX-resistance mutations. Expression of NaV1.6N or NaV1.6A in ND7/LoNav cells generated NaV currents with small, but significant, differences in voltage dependence of activation and inactivation. TTX-resistant versions of both isoforms exhibited significant functional differences compared with the corresponding WT channels. We demonstrated that many of the 15 disease-associated variants studied exhibited isoform-dependent functional effects, and that many of the studied SCN8A variants exhibited functional properties that were not easily classified as either gain- or loss-of-function. Our work illustrates the value of considering molecular and cellular context when investigating SCN8A variants.
Asunto(s)
Empalme Alternativo , Canal de Sodio Activado por Voltaje NAV1.6 , Isoformas de Proteínas , Canal de Sodio Activado por Voltaje NAV1.6/genética , Humanos , Isoformas de Proteínas/genética , Empalme Alternativo/genética , Tetrodotoxina/farmacología , Neuronas/metabolismo , Técnicas de Placa-Clamp , Mutación , Línea Celular , AnimalesRESUMEN
While evolution is often considered from a DNA- and protein-centric view, RNA-based regulation can also impact gene expression and protein sequences. Here we examined interspecies differences in RNA-protein interactions using the conserved neuronal RNA binding protein, Unkempt (UNK) as model. We find that roughly half of mRNAs bound in human are also bound in mouse. Unexpectedly, even when transcript-level binding was conserved across species differential motif usage was prevalent. To understand the biochemical basis of UNK-RNA interactions, we reconstituted the human and mouse UNK-RNA interactomes using a high-throughput biochemical assay. We uncover detailed features driving binding, show that in vivo patterns are captured in vitro, find that highly conserved sites are the strongest bound, and associate binding strength with downstream regulation. Furthermore, subtle sequence differences surrounding motifs are key determinants of species-specific binding. We highlight the complex features driving protein-RNA interactions and how these evolve to confer species-specific regulation.
RESUMEN
Many non-coding variants associated with phenotypes occur in 3' untranslated regions (3' UTRs), and may affect interactions with RNA-binding proteins (RBPs) to regulate gene expression post-transcriptionally. However, identifying functional 3' UTR variants has proven difficult. We use allele frequencies from the Genome Aggregation Database (gnomAD) to identify classes of 3' UTR variants under strong negative selection in humans. We develop intergenic mutability-adjusted proportion singleton (iMAPS), a generalized measure related to MAPS, to quantify negative selection in non-coding regions. This approach, in conjunction with in vitro and in vivo binding data, identifies precise RBP binding sites, miRNA target sites, and polyadenylation signals (PASs) under strong selection. For each class of sites, we identify thousands of gnomAD variants under selection comparable to missense coding variants, and find that sites in core 3' UTR regions upstream of the most-used PAS are under strongest selection. Together, this work improves our understanding of selection on human genes and validates approaches for interpreting genetic variants in human 3' UTRs.
Asunto(s)
MicroARNs , Humanos , Regiones no Traducidas 3'/genética , MicroARNs/genética , MicroARNs/metabolismo , Sitios de Unión/genética , Poliadenilación , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismoRESUMEN
Our ability to determine the clinical impact of variants in 3' untranslated regions (UTRs) of genes remains poor. We provide a thorough analysis of 3' UTR variants from several datasets. Variants in putative regulatory elements, including RNA-binding protein motifs, eCLIP peaks, and microRNA sites, are up to 16 times more likely than variants not in these elements to have gene expression and phenotype associations. Variants in regulatory motifs result in allele-specific protein binding in cell lines and allele-specific gene expression differences in population studies. In addition, variants in shared regions of alternatively polyadenylated isoforms and those proximal to polyA sites are more likely to affect gene expression and phenotype. Finally, pathogenic 3' UTR variants in ClinVar are up to 20 times more likely than benign variants to fall in a regulatory site. We incorporated these findings into RegVar, a software tool that interprets regulatory elements and annotations for any 3' UTR variant and predicts whether the variant is likely to affect gene expression or phenotype. This tool will help prioritize variants for experimental studies and identify pathogenic variants in individuals.
Asunto(s)
MicroARNs , Humanos , Regiones no Traducidas 3'/genética , MicroARNs/genética , Secuencias Reguladoras de Ácidos Nucleicos/genética , Línea Celular , Unión ProteicaRESUMEN
Sequence-specific RNA-binding proteins (RBPs) play central roles in splicing decisions. Here, we describe a modular splicing architecture that leverages in vitro-derived RNA affinity models for 79 human RBPs and the annotated human genome to produce improved models of RBP binding and activity. Binding and activity are modeled by separate Motif and Aggregator components that can be mixed and matched, enforcing sparsity to improve interpretability. Training a new Adjusted Motif (AM) architecture on the splicing task not only yields better splicing predictions but also improves prediction of RBP-binding sites in vivo and of splicing activity, assessed using independent data.
Asunto(s)
Empalme del ARN , Proteínas de Unión al ARN , Humanos , Sitios de Unión , Proteínas de Unión al ARN/metabolismo , ARN/genética , Unión ProteicaRESUMEN
Pathogenic variants in SCN8A , which encodes the voltage-gated sodium (Na V ) channel Na V 1.6, are associated with neurodevelopmental disorders including epileptic encephalopathy. Previous approaches to determine SCN8A variant function may be confounded by the use of a neonatal-expressed alternatively spliced isoform of Na V 1.6 (Na V 1.6N), and engineered mutations to render the channel tetrodotoxin (TTX) resistant. In this study, we investigated the impact of SCN8A alternative splicing on variant function by comparing the functional attributes of 15 variants expressed in two developmentally regulated splice isoforms (Na V 1.6N, Na V 1.6A). We employed automated patch clamp recording to enhance throughput, and developed a novel neuronal cell line (ND7/LoNav) with low levels of endogenous Na V current to obviate the need for TTX-resistance mutations. Expression of Na V 1.6N or Na V 1.6A in ND7/LoNav cells generated Na V currents that differed significantly in voltage-dependence of activation and inactivation. TTX-resistant versions of both isoforms exhibited significant functional differences compared to the corresponding wild-type (WT) channels. We demonstrated that many of the 15 disease-associated variants studied exhibited isoform-dependent functional effects, and that many of the studied SCN8A variants exhibited functional properties that were not easily classified as either gain- or loss-of-function. Our work illustrates the value of considering molecular and cellular context when investigating SCN8A variants.
RESUMEN
Pre-mRNA splicing is surveilled at different stages by quality control (QC) mechanisms. The leukemia-associated DExH-box family helicase hDHX15/scPrp43 is known to disassemble spliceosomes after splicing. Here, using rapid protein depletion and analysis of nascent and mature RNA to enrich for direct effects, we identify a widespread splicing QC function for DHX15 in human cells, consistent with recent in vitro studies. We find that suboptimal introns with weak splice sites, multiple branch points, and cryptic introns are repressed by DHX15, suggesting a general role in promoting splicing fidelity. We identify SUGP1 as a G-patch factor that activates DHX15's splicing QC function. This interaction is dependent on both DHX15's ATPase activity and on SUGP1's U2AF ligand motif (ULM) domain. Together, our results support a model in which DHX15 plays a major role in splicing QC when recruited and activated by SUGP1.
Asunto(s)
Empalme del ARN , Empalmosomas , Humanos , ARN/metabolismo , ARN Helicasas/genética , ARN Helicasas/metabolismo , Precursores del ARN/genética , Precursores del ARN/metabolismo , Empalme del ARN/genética , Factores de Empalme de ARN/genética , Factores de Empalme de ARN/metabolismo , Empalmosomas/metabolismo , Factor de Empalme U2AF/metabolismoRESUMEN
Background: Both promoters and untranslated regions (UTRs) have critical regulatory roles, yet variants in these regions are largely excluded from clinical genetic testing due to difficulty in interpreting pathogenicity. The extent to which these regions may harbour diagnoses for individuals with rare disease is currently unknown. Methods: We present a framework for the identification and annotation of potentially deleterious proximal promoter and UTR variants in known dominant disease genes. We use this framework to annotate de novo variants (DNVs) in 8,040 undiagnosed individuals in the Genomics England 100,000 genomes project, which were subject to strict region-based filtering, clinical review, and validation studies where possible. In addition, we performed region and variant annotation-based burden testing in 7,862 unrelated probands against matched unaffected controls. Results: We prioritised eleven DNVs and identified an additional variant overlapping one of the eleven. Ten of these twelve variants (82%) are in genes that are a strong match to the individual's phenotype and six had not previously been identified. Through burden testing, we did not observe a significant enrichment of potentially deleterious promoter and/or UTR variants in individuals with rare disease collectively across any of our region or variant annotations. Conclusions: Overall, we demonstrate the value of screening promoters and UTRs to uncover additional diagnoses for previously undiagnosed individuals with rare disease and provide a framework for doing so without dramatically increasing interpretation burden.
RESUMEN
Our ability to determine the clinical impact of variants in 3' untranslated regions (UTRs) of genes remains poor. We provide a thorough analysis of 3'UTR variants from several datasets. Variants in putative regulatory elements including RNA-binding protein motifs, eCLIP peaks, and microRNA sites are up to 16 times more likely than other variants to have gene expression and phenotype associations. Heterozygous variants in regulatory motifs result in allele-specific protein binding in cell lines and allele-specific gene expression differences in population studies. In addition, variants in shared regions of alternatively polyadenylated isoforms and those proximal to polyA sites are more likely to affect gene expression and phenotype. Finally, pathogenic 3'UTR variants in ClinVar are 20 times more likely than benign variants to fall in a regulatory site. We incorporated these findings into RegVar, a software tool that interprets regulatory elements and annotations for any 3'UTR variant, and predicts whether the variant is likely to affect gene expression or phenotype. This tool will help prioritize variants for experimental studies and identify pathogenic variants in patients.
RESUMEN
RNA binding proteins (RBPs) are central regulators of gene expression implicated in all facets of RNA metabolism. As such, they play key roles in cellular physiology and disease etiology. Since different steps of post-transcriptional gene expression tend to occur in specific regions of the cell, including nuclear or cytoplasmic locations, defining the subcellular distribution properties of RBPs is an important step in assessing their potential functions. Here, we present the RBP Image Database, a resource that details the subcellular localization features of 301 RBPs in the human HepG2 and HeLa cell lines, based on the results of systematic immuno-fluorescence studies conducted using a highly validated collection of RBP antibodies and a panel of 12 markers for specific organelles and subcellular structures. The unique features of the RBP Image Database include: (i) hosting of comprehensive representative images for each RBP-marker pair, with â¼250,000 microscopy images; (ii) a manually curated controlled vocabulary of annotation terms detailing the localization features of each factor; and (iii) a user-friendly interface allowing the rapid querying of the data by target or annotation. The RBP Image Database is freely available at https://rnabiology.ircm.qc.ca/RBPImage/.
Asunto(s)
Bases de Datos Factuales , Imagen Óptica , Proteínas de Unión al ARN , Humanos , Anticuerpos/metabolismo , Células HeLa , ARN/química , Proteínas de Unión al ARN/metabolismo , Células Hep G2RESUMEN
In breast cancer, nerve presence has been correlated with more invasive disease and worse prognosis, yet the mechanisms by which different types of peripheral nerves drive tumor progression remain poorly understood. In this study, we identified sensory nerves as more abundant in human triple-negative breast cancer (TNBC) tumors. Co-injection of sensory neurons isolated from the dorsal root ganglia (DRG) of adult female mice with human TNBC cells in immunocompromised mice increased the number of lung metastases. Direct in vitro co-culture of human TNBC cells with the dorsal root ganglia (DRG) of adult female mice revealed that TNBC cells adhere to sensory neuron fibers leading to an increase in migration speed. Species-specific RNA sequencing revealed that co-culture of TNBC cells with sensory nerves upregulates the expression of genes associated with cell migration and adhesion in cancer cells. We demonstrated that lack of the semaphorin receptor PlexinB3 in cancer cells attenuate their adhesion to and migration on sensory nerves. Together, our results identify a mechanism by which nerves contribute to breast cancer migration and metastasis by inducing a shift in TNBC cell gene expression and support the rationale for disrupting neuron-cancer cell interactions to target metastasis.
RESUMEN
Messenger RNA isoform differences are predominantly driven by alternative first, internal, and last exons. Despite the importance of classifying exons to understand isoform structure, few tools examine isoform-specific exon usage. We recently observed that alternative transcription start sites often arise near internal exons, often creating "hybrid" first/internal exons. To systematically detect hybrid exons, we built the hybrid-internal-terminal (HIT) pipeline to classify exons depending on their isoform-specific usage. On the basis of splice junction reads in RNA sequencing data and probabilistic modeling, the HIT index identified thousands of previously misclassified hybrid first-internal and internal-last exons. Hybrid exons are enriched in long genes and genes involved in RNA splicing and have longer flanking introns and strong splice sites. Their usage varies considerably across human tissues. By developing the first method to classify exons according to isoform contexts, our findings document the occurrence of hybrid exons, a common quirk of the human transcriptome.
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Empalme Alternativo , Transcriptoma , Secuencia de Bases , Exones , Humanos , Intrones/genéticaRESUMEN
Oxidative phosphorylation (OXPHOS) and glycolysis are the two major pathways for ATP production. The reliance on each varies across tissues and cell states, and can influence susceptibility to disease. At present, the full set of molecular mechanisms governing the relative expression and balance of these two pathways is unknown. Here, we focus on genes whose loss leads to an increase in OXPHOS activity. Unexpectedly, this class of genes is enriched for components of the pre-mRNA splicing machinery, in particular for subunits of the U1 snRNP. Among them, we show that LUC7L2 represses OXPHOS and promotes glycolysis by multiple mechanisms, including (1) splicing of the glycolytic enzyme PFKM to suppress glycogen synthesis, (2) splicing of the cystine/glutamate antiporter SLC7A11 (xCT) to suppress glutamate oxidation, and (3) secondary repression of mitochondrial respiratory supercomplex formation. Our results connect LUC7L2 expression and, more generally, the U1 snRNP to cellular energy metabolism.
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Glucólisis , Fosforilación Oxidativa , Precursores del ARN/metabolismo , Empalme del ARN , ARN Mensajero/metabolismo , Proteínas de Unión al ARN/metabolismo , Ribonucleoproteína Nuclear Pequeña U1/metabolismo , Sistema de Transporte de Aminoácidos y+/genética , Sistema de Transporte de Aminoácidos y+/metabolismo , Proteínas del Complejo de Cadena de Transporte de Electrón/genética , Proteínas del Complejo de Cadena de Transporte de Electrón/metabolismo , Regulación de la Expresión Génica , Estudio de Asociación del Genoma Completo , Ácido Glutámico/metabolismo , Glucógeno/metabolismo , Glucólisis/genética , Células HEK293 , Células HeLa , Humanos , Células K562 , Mitocondrias/genética , Mitocondrias/metabolismo , Oxidación-Reducción , Fosfofructoquinasa-1 Tipo Muscular/genética , Fosfofructoquinasa-1 Tipo Muscular/metabolismo , Precursores del ARN/genética , ARN Mensajero/genética , Proteínas de Unión al ARN/genética , Ribonucleoproteína Nuclear Pequeña U1/genéticaRESUMEN
The Rbfox family of splicing factors regulate alternative splicing during animal development and in disease, impacting thousands of exons in the maturing brain, heart and muscle. Rbfox proteins have long been known to bind to the RNA sequence GCAUG with high affinity and specificity, but just half of Rbfox binding sites contain a GCAUG motif in vivo. We incubated recombinant RBFOX2 with over 60,000 mouse and human transcriptomic sequences to reveal substantial binding to several moderate-affinity, non-GCAYG sites at a physiologically relevant range of RBFOX2 concentrations. We find that these 'secondary motifs' bind Rbfox robustly in cells and that several together can exert regulation comparable to GCAUG in a trichromatic splicing reporter assay. Furthermore, secondary motifs regulate RNA splicing in neuronal development and in neuronal subtypes where cellular Rbfox concentrations are highest, enabling a second wave of splicing changes as Rbfox levels increase.
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Neuronas/fisiología , Factores de Empalme de ARN/química , Factores de Empalme de ARN/metabolismo , Proteínas Represoras/metabolismo , Regiones no Traducidas 3' , Secuencias de Aminoácidos , Sitios de Unión , Antígeno CD47/genética , Antígeno CD47/metabolismo , Diferenciación Celular , Expresión Génica , Células Hep G2 , Humanos , Neuronas/citología , Empalme del ARN , Factores de Empalme de ARN/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteínas Represoras/química , Proteínas Represoras/genética , Análisis de Secuencia de ARNRESUMEN
Many proteins regulate the expression of genes by binding to specific regions encoded in the genome1. Here we introduce a new data set of RNA elements in the human genome that are recognized by RNA-binding proteins (RBPs), generated as part of the Encyclopedia of DNA Elements (ENCODE) project phase III. This class of regulatory elements functions only when transcribed into RNA, as they serve as the binding sites for RBPs that control post-transcriptional processes such as splicing, cleavage and polyadenylation, and the editing, localization, stability and translation of mRNAs. We describe the mapping and characterization of RNA elements recognized by a large collection of human RBPs in K562 and HepG2 cells. Integrative analyses using five assays identify RBP binding sites on RNA and chromatin in vivo, the in vitro binding preferences of RBPs, the function of RBP binding sites and the subcellular localization of RBPs, producing 1,223 replicated data sets for 356 RBPs. We describe the spectrum of RBP binding throughout the transcriptome and the connections between these interactions and various aspects of RNA biology, including RNA stability, splicing regulation and RNA localization. These data expand the catalogue of functional elements encoded in the human genome by the addition of a large set of elements that function at the RNA level by interacting with RBPs.
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Proteínas de Unión al ARN/química , Proteínas de Unión al ARN/metabolismo , Transcriptoma/genética , Empalme Alternativo/genética , Secuencia de Bases , Sitios de Unión , Línea Celular , Cromatina/genética , Cromatina/metabolismo , Bases de Datos Genéticas , Femenino , Técnicas de Silenciamiento del Gen , Humanos , Espacio Intracelular/genética , Masculino , Unión Proteica , ARN Mensajero/química , ARN Mensajero/genética , ARN Mensajero/metabolismo , Proteínas de Unión al ARN/genética , Especificidad por SustratoRESUMEN
The human and mouse genomes contain instructions that specify RNAs and proteins and govern the timing, magnitude, and cellular context of their production. To better delineate these elements, phase III of the Encyclopedia of DNA Elements (ENCODE) Project has expanded analysis of the cell and tissue repertoires of RNA transcription, chromatin structure and modification, DNA methylation, chromatin looping, and occupancy by transcription factors and RNA-binding proteins. Here we summarize these efforts, which have produced 5,992 new experimental datasets, including systematic determinations across mouse fetal development. All data are available through the ENCODE data portal (https://www.encodeproject.org), including phase II ENCODE1 and Roadmap Epigenomics2 data. We have developed a registry of 926,535 human and 339,815 mouse candidate cis-regulatory elements, covering 7.9 and 3.4% of their respective genomes, by integrating selected datatypes associated with gene regulation, and constructed a web-based server (SCREEN; http://screen.encodeproject.org) to provide flexible, user-defined access to this resource. Collectively, the ENCODE data and registry provide an expansive resource for the scientific community to build a better understanding of the organization and function of the human and mouse genomes.
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ADN/genética , Bases de Datos Genéticas , Genoma/genética , Genómica , Anotación de Secuencia Molecular , Sistema de Registros , Secuencias Reguladoras de Ácidos Nucleicos/genética , Animales , Cromatina/genética , Cromatina/metabolismo , ADN/química , Huella de ADN , Metilación de ADN/genética , Momento de Replicación del ADN , Desoxirribonucleasa I/metabolismo , Genoma Humano , Histonas/metabolismo , Humanos , Ratones , Ratones Transgénicos , Proteínas de Unión al ARN/genética , Transcripción Genética/genética , Transposasas/metabolismoRESUMEN
The processing of RNA transcripts from mammalian genes occurs in proximity to their transcription. Here, we describe a phenomenon affecting thousands of genes that we call exon-mediated activation of transcription starts (EMATS), in which the splicing of internal exons impacts promoter choice and the expression level of the gene. We observed that evolutionary gain of internal exons is associated with gain of new transcription start sites (TSSs) nearby and increased gene expression. Inhibiting exon splicing reduced transcription from nearby promoters, and creation of new spliced exons activated transcription from cryptic promoters. The strongest effects occurred for weak promoters located proximal and upstream of efficiently spliced exons. Together, our findings support a model in which splicing recruits transcription machinery locally to influence TSS choice and identify exon gain, loss, and regulatory change as major contributors to the evolution of alternative promoters and gene expression in mammals.