RESUMEN
Host and virus interactions occurring at the post-transcriptional level are critical for infection but remain poorly understood. Here, we performed comprehensive transcriptome-wide analyses revealing that human cytomegalovirus (HCMV) infection results in widespread alternative splicing (AS), shortening of 3' untranslated regions (3' UTRs) and lengthening of poly(A)-tails in host gene transcripts. We found that the host RNA-binding protein CPEB1 was highly induced after infection, and ectopic expression of CPEB1 in noninfected cells recapitulated infection-related post-transcriptional changes. CPEB1 was also required for poly(A)-tail lengthening of viral RNAs important for productive infection. Strikingly, depletion of CPEB1 reversed infection-related cytopathology and post-transcriptional changes, and decreased productive HCMV titers. Host RNA processing was also altered in herpes simplex virus-2 (HSV-2)-infected cells, thereby indicating that this phenomenon might be a common occurrence during herpesvirus infections. We anticipate that our work may serve as a starting point for therapeutic targeting of host RNA-binding proteins in herpesvirus infections.
Asunto(s)
Infecciones por Citomegalovirus/genética , Citomegalovirus/genética , ARN Mensajero/genética , ARN Viral/genética , Factores de Transcripción/genética , Transcriptoma , Factores de Escisión y Poliadenilación de ARNm/genética , Regiones no Traducidas 3' , Empalme Alternativo , Línea Celular , Citomegalovirus/fisiología , Infecciones por Citomegalovirus/metabolismo , Infecciones por Citomegalovirus/patología , Infecciones por Citomegalovirus/virología , Regulación de la Expresión Génica , Interacciones Huésped-Patógeno , Humanos , Poliadenilación , Factores de Transcripción/metabolismo , Regulación hacia Arriba , Factores de Escisión y Poliadenilación de ARNm/metabolismoRESUMEN
RNA metabolism is controlled by an expanding, yet incomplete, catalog of RNA-binding proteins (RBPs), many of which lack characterized RNA binding domains. Approaches to expand the RBP repertoire to discover non-canonical RBPs are currently needed. Here, HaloTag fusion pull down of 12 nuclear and cytoplasmic RBPs followed by quantitative mass spectrometry (MS) demonstrates that proteins interacting with multiple RBPs in an RNA-dependent manner are enriched for RBPs. This motivated SONAR, a computational approach that predicts RNA binding activity by analyzing large-scale affinity precipitation-MS protein-protein interactomes. Without relying on sequence or structure information, SONAR identifies 1,923 human, 489 fly, and 745 yeast RBPs, including over 100 human candidate RBPs that contain zinc finger domains. Enhanced CLIP confirms RNA binding activity and identifies transcriptome-wide RNA binding sites for SONAR-predicted RBPs, revealing unexpected RNA binding activity for disease-relevant proteins and DNA binding proteins.
Asunto(s)
Algoritmos , Anotación de Secuencia Molecular , Proteínas de Unión al ARN/química , Proteínas de Unión al ARN/clasificación , ARN/química , Animales , Sitios de Unión , Núcleo Celular/química , Núcleo Celular/metabolismo , Citoplasma/química , Citoplasma/metabolismo , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Expresión Génica , Ontología de Genes , Células HEK293 , Humanos , Motivos de Nucleótidos , Unión Proteica , Dominios y Motivos de Interacción de Proteínas , ARN/genética , ARN/metabolismo , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Programas Informáticos , Dedos de ZincRESUMEN
HnRNPA2B1 encodes an RNA binding protein associated with neurodegeneration. However, its function in the nervous system is unclear. Transcriptome-wide crosslinking and immunoprecipitation in mouse spinal cord discover UAGG motifs enriched within â¼2,500 hnRNP A2/B1 binding sites and an unexpected role for hnRNP A2/B1 in alternative polyadenylation. HnRNP A2/B1 loss results in alternative splicing (AS), including skipping of an exon in amyotrophic lateral sclerosis (ALS)-associated D-amino acid oxidase (DAO) that reduces D-serine metabolism. ALS-associated hnRNP A2/B1 D290V mutant patient fibroblasts and motor neurons differentiated from induced pluripotent stem cells (iPSC-MNs) demonstrate abnormal splicing changes, likely due to increased nuclear-insoluble hnRNP A2/B1. Mutant iPSC-MNs display decreased survival in long-term culture and exhibit hnRNP A2/B1 localization to cytoplasmic granules as well as exacerbated changes in gene expression and splicing upon cellular stress. Our findings provide a cellular resource and reveal RNA networks relevant to neurodegeneration, regulated by normal and mutant hnRNP A2/B1. VIDEO ABSTRACT.
Asunto(s)
Empalme Alternativo/genética , Esclerosis Amiotrófica Lateral/genética , Supervivencia Celular/genética , Fibroblastos/metabolismo , Ribonucleoproteína Heterogénea-Nuclear Grupo A-B/genética , Neuronas Motoras/metabolismo , Transporte de Proteínas/genética , Esclerosis Amiotrófica Lateral/metabolismo , Animales , Estudios de Casos y Controles , D-Aminoácido Oxidasa/genética , D-Aminoácido Oxidasa/metabolismo , Técnica del Anticuerpo Fluorescente , Expresión Génica , Perfilación de la Expresión Génica , Ribonucleoproteína Heterogénea-Nuclear Grupo A-B/metabolismo , Humanos , Células Madre Pluripotentes Inducidas , Ratones , Mutación , PoliadenilaciónRESUMEN
The RNA-binding protein (RBP) TAF15 is implicated in amyotrophic lateral sclerosis (ALS). To compare TAF15 function to that of two ALS-associated RBPs, FUS and TDP-43, we integrate CLIP-seq and RNA Bind-N-Seq technologies, and show that TAF15 binds to â¼4,900 RNAs enriched for GGUA motifs in adult mouse brains. TAF15 and FUS exhibit similar binding patterns in introns, are enriched in 3' untranslated regions and alter genes distinct from TDP-43. However, unlike FUS and TDP-43, TAF15 has a minimal role in alternative splicing. In human neural progenitors, TAF15 and FUS affect turnover of their RNA targets. In human stem cell-derived motor neurons, the RNA profile associated with concomitant loss of both TAF15 and FUS resembles that observed in the presence of the ALS-associated mutation FUS R521G, but contrasts with late-stage sporadic ALS patients. Taken together, our findings reveal convergent and divergent roles for FUS, TAF15 and TDP-43 in RNA metabolism.
Asunto(s)
Empalme Alternativo/genética , Esclerosis Amiotrófica Lateral/genética , Proteínas de Unión al ADN/genética , Proteína FUS de Unión a ARN/genética , Factores Asociados con la Proteína de Unión a TATA/genética , Regiones no Traducidas 3'/genética , Animales , Biología Computacional/métodos , Proteínas de Unión al ADN/metabolismo , Modelos Animales de Enfermedad , Femenino , Fibroblastos , Técnicas de Silenciamiento del Gen , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Humanos , Células Madre Pluripotentes Inducidas , Intrones/genética , Ratones , Ratones Endogámicos C57BL , Neuronas Motoras/metabolismo , Mutación , Oligonucleótidos Antisentido/administración & dosificación , Oligonucleótidos Antisentido/genética , Cultivo Primario de Células , ARN Mensajero/genética , ARN Mensajero/metabolismo , ARN Interferente Pequeño/metabolismo , Proteína FUS de Unión a ARN/metabolismo , Análisis de Secuencia de ARN/métodos , Factores Asociados con la Proteína de Unión a TATA/metabolismoRESUMEN
After an RNA is transcribed, it undergoes a variety of processing steps that can change the encoded protein sequence (through alternative splicing and RNA editing), regulate the stability of the RNA, and control subcellular localization, timing, and rate of translation. The recent explosion in genomics techniques has enabled transcriptome-wide profiling of RNA processing in an unbiased manner. However, it has also brought with it both experimental challenges in developing improved methods to probe distinct processing steps, as well as computational challenges in data storage, processing, and analysis tools to enable large-scale interpretation in the genomics era. In this chapter we review experimental techniques and challenges in profiling various aspects of RNA processing, as well as recent efforts to develop analyses integrating multiple data sources and techniques to infer RNA regulatory networks.
Asunto(s)
Biología Computacional/métodos , Proteínas de Unión al ARN/metabolismo , ARN/metabolismo , Animales , Sitios de Unión , Conjuntos de Datos como Asunto , Perfilación de la Expresión Génica/métodos , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Humanos , Inmunoprecipitación , Modelos Moleculares , Mutación , Conformación de Ácido Nucleico , Unión Proteica , Conformación Proteica , Precursores del ARN/metabolismo , Procesamiento Postranscripcional del ARN , Empalme del ARN , Estabilidad del ARN , Motivos de Unión al ARN , Análisis de Secuencia de ARNRESUMEN
Transcriptome-wide maps of RNA binding protein (RBP)-RNA interactions by immunoprecipitation (IP)-based methods such as RNA IP (RIP) and crosslinking and IP (CLIP) are key starting points for evaluating the molecular roles of the thousands of human RBPs. A significant bottleneck to the application of these methods in diverse cell lines, tissues, and developmental stages is the availability of validated IP-quality antibodies. Using IP followed by immunoblot assays, we have developed a validated repository of 438 commercially available antibodies that interrogate 365 unique RBPs. In parallel, 362 short-hairpin RNA (shRNA) constructs against 276 unique RBPs were also used to confirm specificity of these antibodies. These antibodies can characterize subcellular RBP localization. With the burgeoning interest in the roles of RBPs in cancer, neurobiology, and development, these resources are invaluable to the broad scientific community. Detailed information about these resources is publicly available at the ENCODE portal (https://www.encodeproject.org/).
Asunto(s)
Bases de Datos Genéticas , Proteínas de Unión al ARN/genética , ARN/metabolismo , Transcriptoma/genética , Sitios de Unión , Humanos , Unión Proteica , ARN/genética , ARN Interferente Pequeño/clasificación , ARN Interferente Pequeño/genética , Proteínas de Unión al ARN/metabolismoRESUMEN
Fusion genes are known to be key drivers of tumor growth in several types of cancer. Traditionally, detecting fusion genes has been a difficult task based on fluorescent in situ hybridization to detect chromosomal abnormalities. More recently, RNA sequencing has enabled an increased pace of fusion gene identification. However, RNA-Seq is inefficient for the identification of fusion genes due to the high number of sequencing reads needed to detect the small number of fusion transcripts present in cells of interest. Here we describe a method, Single Primer Enrichment Technology (SPET), for targeted RNA sequencing that is customizable to any target genes, is simple to use, and efficiently detects gene fusions. Using SPET to target 5701 exons of 401 known cancer fusion genes for sequencing, we were able to identify known and previously unreported gene fusions from both fresh-frozen and formalin-fixed paraffin-embedded (FFPE) tissue RNA in both normal tissue and cancer cells.
Asunto(s)
Fusión Génica , Secuenciación de Nucleótidos de Alto Rendimiento , Hibridación Fluorescente in Situ , ARN , Neoplasias de la Mama/genética , Neoplasias de la Mama/patología , Carcinoma Ductal de Mama/genética , Carcinoma Ductal de Mama/patología , Humanos , Fusión de Oncogenes , ARN/genéticaRESUMEN
Transactivating response region DNA binding protein (TDP-43) is the major protein component of ubiquitinated inclusions found in amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD) with ubiquitinated inclusions. Two ALS-causing mutants (TDP-43(Q331K) and TDP-43(M337V)), but not wild-type human TDP-43, are shown here to provoke age-dependent, mutant-dependent, progressive motor axon degeneration and motor neuron death when expressed in mice at levels and in a cell type-selective pattern similar to endogenous TDP-43. Mutant TDP-43-dependent degeneration of lower motor neurons occurs without: (i) loss of TDP-43 from the corresponding nuclei, (ii) accumulation of TDP-43 aggregates, and (iii) accumulation of insoluble TDP-43. Computational analysis using splicing-sensitive microarrays demonstrates alterations of endogenous TDP-43-dependent alternative splicing events conferred by both human wild-type and mutant TDP-43(Q331K), but with high levels of mutant TDP-43 preferentially enhancing exon exclusion of some target pre-mRNAs affecting genes involved in neurological transmission and function. Comparison with splicing alterations following TDP-43 depletion demonstrates that TDP-43(Q331K) enhances normal TDP-43 splicing function for some RNA targets but loss-of-function for others. Thus, adult-onset motor neuron disease does not require aggregation or loss of nuclear TDP-43, with ALS-linked mutants producing loss and gain of splicing function of selected RNA targets at an early disease stage.
Asunto(s)
Esclerosis Amiotrófica Lateral/genética , Núcleo Celular/metabolismo , Proteínas de Unión al ADN/genética , Mutación , Empalme del ARN , Esclerosis Amiotrófica Lateral/metabolismo , Esclerosis Amiotrófica Lateral/fisiopatología , Animales , Proteínas de Unión al ADN/metabolismo , Ratones , Ratones Transgénicos , Reacción en Cadena en Tiempo Real de la Polimerasa , UbiquitinaciónRESUMEN
FUS/TLS (fused in sarcoma/translocated in liposarcoma) and TDP-43 are integrally involved in amyotrophic lateral sclerosis (ALS) and frontotemporal dementia. We found that FUS/TLS binds to RNAs from >5,500 genes in mouse and human brain, primarily through a GUGGU-binding motif. We identified a sawtooth-like binding pattern, consistent with co-transcriptional deposition of FUS/TLS. Depletion of FUS/TLS from the adult nervous system altered the levels or splicing of >950 mRNAs, most of which are distinct from RNAs dependent on TDP-43. Abundance of only 45 RNAs was reduced after depletion of either TDP-43 or FUS/TLS from mouse brain, but among these were mRNAs that were transcribed from genes with exceptionally long introns and that encode proteins that are essential for neuronal integrity. Expression levels of a subset of these were lowered after TDP-43 or FUS/TLS depletion in stem cell-derived human neurons and in TDP-43 aggregate-containing motor neurons in sporadic ALS, supporting a common loss-of-function pathway as one component underlying motor neuron death from misregulation of TDP-43 or FUS/TLS.
Asunto(s)
Esclerosis Amiotrófica Lateral/metabolismo , Proteínas de Unión al ADN/metabolismo , Demencia Frontotemporal/metabolismo , Precursores del ARN/metabolismo , ARN Mensajero/metabolismo , Proteína FUS de Unión a ARN/metabolismo , Proteínas Adaptadoras Transductoras de Señales , Esclerosis Amiotrófica Lateral/genética , Esclerosis Amiotrófica Lateral/patología , Animales , Proteínas Relacionadas con la Autofagia , Encéfalo/metabolismo , Encéfalo/patología , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Línea Celular Transformada , Proteínas de Unión al ADN/deficiencia , Proteínas de Unión al ADN/genética , Transportador 2 de Aminoácidos Excitadores/genética , Transportador 2 de Aminoácidos Excitadores/metabolismo , Femenino , Demencia Frontotemporal/genética , Demencia Frontotemporal/patología , Perfilación de la Expresión Génica , Regulación de la Expresión Génica/genética , N-Metiltransferasa de Histona-Lisina/metabolismo , Humanos , Inmunoprecipitación , Proteínas de Interacción con los Canales Kv/metabolismo , Proteínas de la Membrana/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Neuronas Motoras/metabolismo , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , Moléculas de Adhesión de Célula Nerviosa/metabolismo , Células-Madre Neurales/metabolismo , Proteínas de Neurofilamentos/metabolismo , Análisis de Secuencia por Matrices de Oligonucleótidos , Unión Proteica/genética , Estructura Terciaria de Proteína/genética , Precursores del ARN/genética , Empalme del ARN/genética , ARN Mensajero/genética , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Proteína FUS de Unión a ARN/deficiencia , Proteína FUS de Unión a ARN/genética , Canales de Potasio Shal/metabolismo , Médula Espinal/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Proteínas tau/genética , Proteínas tau/metabolismoRESUMEN
LIN28 is a conserved RNA-binding protein implicated in pluripotency, reprogramming, and oncogenesis. It was previously shown to act primarily by blocking let-7 microRNA (miRNA) biogenesis, but here we elucidate distinct roles of LIN28 regulation via its direct messenger RNA (mRNA) targets. Through crosslinking and immunoprecipitation coupled with high-throughput sequencing (CLIP-seq) in human embryonic stem cells and somatic cells expressing exogenous LIN28, we have defined discrete LIN28-binding sites in a quarter of human transcripts. These sites revealed that LIN28 binds to GGAGA sequences enriched within loop structures in mRNAs, reminiscent of its interaction with let-7 miRNA precursors. Among LIN28 mRNA targets, we found evidence for LIN28 autoregulation and also direct but differing effects on the protein abundance of splicing regulators in somatic and pluripotent stem cells. Splicing-sensitive microarrays demonstrated that exogenous LIN28 expression causes widespread downstream alternative splicing changes. These findings identify important regulatory functions of LIN28 via direct mRNA interactions.
Asunto(s)
Empalme Alternativo/genética , ARN Mensajero , Proteínas de Unión al ARN , Sitios de Unión/genética , Células Madre Embrionarias , Regulación del Desarrollo de la Expresión Génica , Células HEK293 , Humanos , Motivos de Nucleótidos , ARN Mensajero/genética , ARN Mensajero/metabolismo , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismoRESUMEN
Understanding how RNA binding proteins control the splicing code is fundamental to human biology and disease. Here, we present a comprehensive study to elucidate how heterogeneous nuclear ribonucleoparticle (hnRNP) proteins, among the most abundant RNA binding proteins, coordinate to regulate alternative pre-mRNA splicing (AS) in human cells. Using splicing-sensitive microarrays, crosslinking and immunoprecipitation coupled with high-throughput sequencing (CLIP-seq), and cDNA sequencing, we find that more than half of all AS events are regulated by multiple hnRNP proteins and that some combinations of hnRNP proteins exhibit significant synergy, whereas others act antagonistically. Our analyses reveal position-dependent RNA splicing maps, in vivo consensus binding sites, a surprising level of cross- and autoregulation among hnRNP proteins, and the coordinated regulation by hnRNP proteins of dozens of other RNA binding proteins and genes associated with cancer. Our findings define an unprecedented degree of complexity and compensatory relationships among hnRNP proteins and their splicing targets that likely confer robustness to cells.
Asunto(s)
Empalme Alternativo/genética , Genoma Humano/genética , Ribonucleoproteínas Nucleares Heterogéneas/metabolismo , Secuencia de Bases , Sitios de Unión/genética , Western Blotting , Exones/genética , Fibroblastos/metabolismo , Genes Relacionados con las Neoplasias/genética , Células HEK293 , Humanos , Datos de Secuencia Molecular , Motivos de Nucleótidos/genética , Análisis de Secuencia por Matrices de Oligonucleótidos , Especificidad de Órganos/genética , Unión Proteica/genética , Mapeo de Interacción de Proteínas , Precursores del ARN/metabolismo , Reproducibilidad de los Resultados , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
We used cross-linking and immunoprecipitation coupled with high-throughput sequencing to identify binding sites in 6,304 genes as the brain RNA targets for TDP-43, an RNA binding protein that, when mutated, causes amyotrophic lateral sclerosis. Massively parallel sequencing and splicing-sensitive junction arrays revealed that levels of 601 mRNAs were changed (including Fus (Tls), progranulin and other transcripts encoding neurodegenerative disease-associated proteins) and 965 altered splicing events were detected (including in sortilin, the receptor for progranulin) following depletion of TDP-43 from mouse adult brain with antisense oligonucleotides. RNAs whose levels were most depleted by reduction in TDP-43 were derived from genes with very long introns and that encode proteins involved in synaptic activity. Lastly, we found that TDP-43 autoregulates its synthesis, in part by directly binding and enhancing splicing of an intron in the 3' untranslated region of its own transcript, thereby triggering nonsense-mediated RNA degradation.
Asunto(s)
Empalme Alternativo/genética , Esclerosis Amiotrófica Lateral/genética , Proteínas de Unión al ADN/genética , Degeneración Nerviosa/genética , Neuronas/patología , Precursores del ARN/genética , ARN Mensajero/genética , Regiones no Traducidas 3'/genética , Esclerosis Amiotrófica Lateral/metabolismo , Esclerosis Amiotrófica Lateral/fisiopatología , Animales , Proteínas de Unión al ADN/biosíntesis , Proteínas de Unión al ADN/deficiencia , Femenino , Homeostasis/genética , Humanos , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Degeneración Nerviosa/metabolismo , Degeneración Nerviosa/fisiopatología , Neuronas/metabolismo , Oligonucleótidos Antisentido/genética , Precursores del ARN/antagonistas & inhibidores , ARN Mensajero/antagonistas & inhibidoresRESUMEN
The structure of the K262R genetic variant of human cytochrome P450 2B6 in complex with the inhibitor 4-(4-chlorophenyl)imidazole (4-CPI) has been determined using X-ray crystallography to 2.0-A resolution. Production of diffraction quality crystals was enabled through a combination of protein engineering, chaperone coexpression, modifications to the purification protocol, and the use of unique facial amphiphiles during crystallization. The 2B6-4-CPI complex is virtually identical to the rabbit 2B4 structure bound to the same inhibitor with respect to the arrangement of secondary structural elements and the placement of active site residues. The structure supports prior P450 2B6 homology models based on other mammalian cytochromes P450 and is consistent with the limited site-directed mutagenesis studies on 2B6 and extensive studies on P450 2B4 and 2B1. Although the K262R genetic variant shows unaltered binding of 4-CPI, altered binding affinity, kinetics, and/or product profiles have been previously shown with several other ligands. On the basis of new P450 2B6 crystal structure and previous 2B4 structures, substitutions at residue 262 affect a hydrogen-bonding network connecting the G and H helices, where subtle differences could be transduced to the active site. Docking experiments indicate that the closed protein conformation allows smaller ligands such as ticlopidine to bind to the 2B6 active site in the expected orientation. However, it is unknown whether 2B6 undergoes structural reorganization to accommodate bulkier molecules, as previously inferred from multiple P450 2B4 crystal structures.