RESUMEN
The twenty-three Fanconi anemia (FA) proteins cooperate in the FA/BRCA pathway to repair DNA interstrand cross-links (ICLs). The cell division cycle and apoptosis regulator 1 (CCAR1) protein is also a regulator of ICL repair, though its possible function in the FA/BRCA pathway remains unknown. Here, we demonstrate that CCAR1 plays a unique upstream role in the FA/BRCA pathway and is required for FANCA protein expression in human cells. Interestingly, CCAR1 co-immunoprecipitates with FANCA pre-mRNA and is required for FANCA mRNA processing. Loss of CCAR1 results in retention of a poison exon in the FANCA transcript, thereby leading to reduced FANCA protein expression. A unique domain of CCAR1, the EF hand domain, is required for interaction with the U2AF heterodimer of the spliceosome and for excision of the poison exon. Taken together, CCAR1 is a splicing modulator required for normal splicing of the FANCA mRNA and other mRNAs involved in various cellular pathways.
Asunto(s)
Proteínas Reguladoras de la Apoptosis , Proteínas de Ciclo Celular , Proteína del Grupo de Complementación A de la Anemia de Fanconi , Anemia de Fanconi , Empalme del ARN , Factor de Empalme U2AF , Humanos , Proteína BRCA1/metabolismo , Proteína BRCA1/genética , Proteína BRCA2/metabolismo , Proteína BRCA2/genética , Reparación del ADN , Endodesoxirribonucleasas , Exones , Anemia de Fanconi/genética , Anemia de Fanconi/metabolismo , Proteína del Grupo de Complementación A de la Anemia de Fanconi/genética , Proteína del Grupo de Complementación A de la Anemia de Fanconi/metabolismo , Células HEK293 , Células HeLa , Unión Proteica , Precursores del ARN/metabolismo , Precursores del ARN/genética , ARN Mensajero/genética , ARN Mensajero/metabolismo , Transducción de Señal , Empalmosomas/metabolismo , Empalmosomas/genética , Factor de Empalme U2AF/metabolismo , Factor de Empalme U2AF/genética , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Proteínas Reguladoras de la Apoptosis/genética , Proteínas Reguladoras de la Apoptosis/metabolismoRESUMEN
Huntington's disease (HD) is a fatal, dominantly inherited neurodegenerative disorder caused by CAG trinucleotide expansion in exon 1 of the huntingtin (HTT) gene. Since the reduction of pathogenic mutant HTT messenger RNA is therapeutic, we developed a mutant allele-sensitive CAGEX RNA-targeting CRISPR-Cas13d system (Cas13d-CAGEX) that eliminates toxic CAGEX RNA in fibroblasts derived from patients with HD and induced pluripotent stem cell-derived neurons. We show that intrastriatal delivery of Cas13d-CAGEX via an adeno-associated viral vector selectively reduces mutant HTT mRNA and protein levels in the striatum of heterozygous zQ175 mice, a model of HD. This also led to improved motor coordination, attenuated striatal atrophy and reduction of mutant HTT protein aggregates. These phenotypic improvements lasted for at least eight months without adverse effects and with minimal off-target transcriptomic effects. Taken together, we demonstrate proof of principle of an RNA-targeting CRISPR-Cas13d system as a therapeutic approach for HD, a strategy with implications for the treatment of other dominantly inherited disorders.
Asunto(s)
Enfermedad de Huntington , Ratones , Animales , Enfermedad de Huntington/genética , Enfermedad de Huntington/terapia , Enfermedad de Huntington/metabolismo , ARN , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas , Cuerpo Estriado/metabolismo , ARN Mensajero/metabolismo , Fenotipo , Proteína Huntingtina/genética , Proteína Huntingtina/metabolismo , Modelos Animales de EnfermedadRESUMEN
RNA-binding proteins (RBPs) are critical regulators of gene expression and RNA processing that are required for gene function. Yet the dynamics of RBP regulation in single cells is unknown. To address this gap in understanding, we developed STAMP (Surveying Targets by APOBEC-Mediated Profiling), which efficiently detects RBP-RNA interactions. STAMP does not rely on ultraviolet cross-linking or immunoprecipitation and, when coupled with single-cell capture, can identify RBP-specific and cell-type-specific RNA-protein interactions for multiple RBPs and cell types in single, pooled experiments. Pairing STAMP with long-read sequencing yields RBP target sites in an isoform-specific manner. Finally, Ribo-STAMP leverages small ribosomal subunits to measure transcriptome-wide ribosome association in single cells. STAMP enables the study of RBP-RNA interactomes and translational landscapes with unprecedented cellular resolution.
Asunto(s)
Proteínas de Unión al ARN/metabolismo , ARN/metabolismo , Análisis de la Célula Individual/métodos , Animales , Sitios de Unión , Perfilación de la Expresión Génica , Células HEK293 , Humanos , Secuenciación de Nanoporos , ARN/química , Proteínas de Unión al ARN/química , Análisis de Secuencia de ARN , TranscriptomaRESUMEN
Site-directed RNA editing approaches offer great potential to correct genetic mutations in somatic cells while avoiding permanent off-target genomic edits. Nuclease-dead RNA-targeting CRISPR-Cas systems recruit functional effectors to RNA molecules in a programmable fashion. Here, we demonstrate a Streptococcus pyogenes Cas9-ADAR2 fusion system that uses a 3' modified guide RNA (gRNA) to enable adenosine-to-inosine (A-to-I) editing of specific bases on reporter and endogenously expressed mRNAs. Due to the sufficient nature of the 3' gRNA extension sequence, we observe that Cas9 gRNA spacer sequences are dispensable for directed RNA editing, revealing that Cas9 can act as an RNA-aptamer-binding protein. We demonstrate that Cas9-based A-to-I editing is comparable in on-target efficiency and off-target specificity with Cas13 RNA editing versions. This study provides a systematic benchmarking of RNA-targeting CRISPR-Cas designs for reversible nucleotide-level conversion at the transcriptome level.
Asunto(s)
Sistemas CRISPR-Cas/genética , Ingeniería Genética , Edición de ARN/genética , Adenosina Desaminasa/metabolismo , Secuencia de Bases , Proteína 9 Asociada a CRISPR/metabolismo , Células HEK293 , Humanos , ARN Guía de Kinetoplastida/metabolismo , Proteínas de Unión al ARN/metabolismo , RNA-Seq , Transcriptoma/genéticaRESUMEN
Microsatellite repeat expansions in DNA produce pathogenic RNA species that cause dominantly inherited diseases such as myotonic dystrophy type 1 and 2 (DM1/2), Huntington's disease, and C9orf72-linked amyotrophic lateral sclerosis (C9-ALS). Means to target these repetitive RNAs are required for diagnostic and therapeutic purposes. Here, we describe the development of a programmable CRISPR system capable of specifically visualizing and eliminating these toxic RNAs. We observe specific targeting and efficient elimination of microsatellite repeat expansion RNAs both when exogenously expressed and in patient cells. Importantly, RNA-targeting Cas9 (RCas9) reverses hallmark features of disease including elimination of RNA foci among all conditions studied (DM1, DM2, C9-ALS, polyglutamine diseases), reduction of polyglutamine protein products, relocalization of repeat-bound proteins to resemble healthy controls, and efficient reversal of DM1-associated splicing abnormalities in patient myotubes. Finally, we report a truncated RCas9 system compatible with adeno-associated viral packaging. This effort highlights the potential of RCas9 for human therapeutics.
Asunto(s)
Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas , Terapia Genética/métodos , Oligonucleótidos Antisentido/farmacología , Animales , Células COS , Línea Celular , Células Cultivadas , Chlorocebus aethiops , Repeticiones de Microsatélite , Empalme del ARN , Expansión de Repetición de TrinucleótidoRESUMEN
Millions of cis-regulatory elements are predicted to be present in the human genome, but direct evidence for their biological function is scarce. Here we report a high-throughput method, cis-regulatory element scan by tiling-deletion and sequencing (CREST-seq), for the unbiased discovery and functional assessment of cis-regulatory sequences in the genome. We used it to interrogate the 2-Mb POU5F1 locus in human embryonic stem cells, and identified 45 cis-regulatory elements. A majority of these elements have active chromatin marks, DNase hypersensitivity, and occupancy by multiple transcription factors, which confirms the utility of chromatin signatures in cis-element mapping. Notably, 17 of them are previously annotated promoters of functionally unrelated genes, and like typical enhancers, they form extensive spatial contacts with the POU5F1 promoter. These results point to the commonality of enhancer-like promoters in the human genome.
Asunto(s)
Mapeo Cromosómico/métodos , Pruebas Genéticas/métodos , Secuencias Reguladoras de Ácidos Nucleicos/genética , Algoritmos , Células Cultivadas , Células Madre Embrionarias/fisiología , Regulación de la Expresión Génica/genética , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Análisis de Secuencia de ADN , Análisis de la Célula IndividualRESUMEN
Intragenic 5-methylcytosine and CTCF mediate opposing effects on pre-mRNA splicing: CTCF promotes inclusion of weak upstream exons through RNA polymerase II pausing, whereas 5-methylcytosine evicts CTCF, leading to exon exclusion. However, the mechanisms governing dynamic DNA methylation at CTCF-binding sites were unclear. Here, we reveal the methylcytosine dioxygenases TET1 and TET2 as active regulators of CTCF-mediated alternative splicing through conversion of 5-methylcytosine to its oxidation derivatives. 5-hydroxymethylcytosine and 5-carboxylcytosine are enriched at an intragenic CTCF-binding sites in the CD45 model gene and are associated with alternative exon inclusion. Reduced TET levels culminate in increased 5-methylcytosine, resulting in CTCF eviction and exon exclusion. In vitro analyses establish the oxidation derivatives are not sufficient to stimulate splicing, but efficiently promote CTCF association. We further show genomewide that reciprocal exchange of 5-hydroxymethylcytosine and 5-methylcytosine at downstream CTCF-binding sites is a general feature of alternative splicing in naïve and activated CD4(+) T cells. These findings significantly expand our current concept of the pre-mRNA "splicing code" to include dynamic intragenic DNA methylation catalyzed by the TET proteins.