RESUMEN
RNA-binding proteins (RBPs) control RNA metabolism to orchestrate gene expression and, when dysfunctional, underlie human diseases. Proteome-wide discovery efforts predict thousands of RBP candidates, many of which lack canonical RNA-binding domains (RBDs). Here, we present a hybrid ensemble RBP classifier (HydRA), which leverages information from both intermolecular protein interactions and internal protein sequence patterns to predict RNA-binding capacity with unparalleled specificity and sensitivity using support vector machines (SVMs), convolutional neural networks (CNNs), and Transformer-based protein language models. Occlusion mapping by HydRA robustly detects known RBDs and predicts hundreds of uncharacterized RNA-binding associated domains. Enhanced CLIP (eCLIP) for HydRA-predicted RBP candidates reveals transcriptome-wide RNA targets and confirms RNA-binding activity for HydRA-predicted RNA-binding associated domains. HydRA accelerates construction of a comprehensive RBP catalog and expands the diversity of RNA-binding associated domains.
Asunto(s)
Aprendizaje Profundo , Hydra , Animales , Humanos , ARN/metabolismo , Unión Proteica , Sitios de Unión/genética , Hydra/genética , Hydra/metabolismoRESUMEN
The cell is a multi-scale structure with modular organization across at least four orders of magnitude1. Two central approaches for mapping this structure-protein fluorescent imaging and protein biophysical association-each generate extensive datasets, but of distinct qualities and resolutions that are typically treated separately2,3. Here we integrate immunofluorescence images in the Human Protein Atlas4 with affinity purifications in BioPlex5 to create a unified hierarchical map of human cell architecture. Integration is achieved by configuring each approach as a general measure of protein distance, then calibrating the two measures using machine learning. The map, known as the multi-scale integrated cell (MuSIC 1.0), resolves 69 subcellular systems, of which approximately half are to our knowledge undocumented. Accordingly, we perform 134 additional affinity purifications and validate subunit associations for the majority of systems. The map reveals a pre-ribosomal RNA processing assembly and accessory factors, which we show govern rRNA maturation, and functional roles for SRRM1 and FAM120C in chromatin and RPS3A in splicing. By integration across scales, MuSIC increases the resolution of imaging while giving protein interactions a spatial dimension, paving the way to incorporate diverse types of data in proteome-wide cell maps.
Asunto(s)
Cromosomas , Proteoma , Antígenos Nucleares/genética , Antígenos Nucleares/metabolismo , Cromatina/genética , Cromosomas/metabolismo , Humanos , Proteínas Asociadas a Matriz Nuclear/metabolismo , Proteoma/metabolismo , ARN Ribosómico , Proteínas de Unión al ARN/genéticaRESUMEN
Skeletal muscle atrophy is a morbidity and mortality risk factor that happens with disuse, chronic disease, and aging. The tissue remodeling that happens during recovery from atrophy or injury involves changes in different cell types such as muscle fibers, and satellite and immune cells. Here, we show that the previously uncharacterized gene and protein Zfp697 is a damage-induced regulator of muscle remodeling. Zfp697/ZNF697 expression is transiently elevated during recovery from muscle atrophy or injury in mice and humans. Sustained Zfp697 expression in mouse muscle leads to a gene expression signature of chemokine secretion, immune cell recruitment, and extracellular matrix remodeling. Notably, although Zfp697 is expressed in several cell types in skeletal muscle, myofiber-specific Zfp697 genetic ablation in mice is sufficient to hinder the inflammatory and regenerative response to muscle injury, compromising functional recovery. We show that Zfp697 is an essential mediator of the interferon gamma response in muscle cells and that it functions primarily as an RNA-interacting protein, with a very high number of miRNA targets. This work identifies Zfp697 as an integrator of cell-cell communication necessary for tissue remodeling and regeneration.
Asunto(s)
Músculo Esquelético , Proteínas de Unión al ARN , Animales , Ratones , Músculo Esquelético/metabolismo , Músculo Esquelético/patología , Proteínas de Unión al ARN/metabolismo , Proteínas de Unión al ARN/genética , Humanos , Inflamación/metabolismo , Inflamación/patología , Inflamación/genética , Ratones Noqueados , Atrofia Muscular/metabolismo , Atrofia Muscular/genética , Atrofia Muscular/patología , MicroARNs/genética , MicroARNs/metabolismo , Ratones Endogámicos C57BL , Interferón gamma/metabolismoRESUMEN
RNA-binding proteins (RBPs) modulate alternative splicing outcomes to determine isoform expression and cellular survival. To identify RBPs that directly drive alternative exon inclusion, we developed tethered function luciferase-based splicing reporters that provide rapid, scalable and robust readouts of exon inclusion changes and used these to evaluate 718 human RBPs. We performed enhanced cross-linking immunoprecipitation, RNA sequencing and affinity purification-mass spectrometry to investigate a subset of candidates with no prior association with splicing. Integrative analysis of these assays indicates surprising roles for TRNAU1AP, SCAF8 and RTCA in the modulation of hundreds of endogenous splicing events. We also leveraged our tethering assays and top candidates to identify potent and compact exon inclusion activation domains for splicing modulation applications. Using these identified domains, we engineered programmable fusion proteins that outperform current artificial splicing factors at manipulating inclusion of reporter and endogenous exons. This tethering approach characterizes the ability of RBPs to induce exon inclusion and yields new molecular parts for programmable splicing control.
RESUMEN
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
Muscular atrophy is a mortality risk factor that happens with disuse, chronic disease, and aging. Recovery from atrophy requires changes in several cell types including muscle fibers, and satellite and immune cells. Here we show that Zfp697/ZNF697 is a damage-induced regulator of muscle regeneration, during which its expression is transiently elevated. Conversely, sustained Zfp697 expression in mouse muscle leads to a gene expression signature of chemokine secretion, immune cell recruitment, and extracellular matrix remodeling. Myofiber-specific Zfp697 ablation hinders the inflammatory and regenerative response to muscle injury, compromising functional recovery. We uncover Zfp697 as an essential interferon gamma mediator in muscle cells, interacting primarily with ncRNAs such as the pro-regenerative miR-206. In sum, we identify Zfp697 as an integrator of cell-cell communication necessary for tissue regeneration.
RESUMEN
Chemical-peptide conjugation is the molecular initiating event in skin sensitization. The OECD test guideline uses a high-performance liquid chromatography/ultraviolet (HPLC/UV) detection method to quantify chemical-peptide conjugation in a direct peptide reactivity assay (DPRA), which measures the depletion of two synthetic peptides containing lysine or cysteine residues. To improve assay throughput, sensitivity and accuracy, an automated 384-well plate-based RapidFire solid-phase extraction (SPE) system coupled with tandem mass spectrometry (MS/MS) DPRA was developed and validated in the presence of a newly designed internal standard. Compared to the HPLC/UV-based DPRA, the automated SPE-MS/MS-based DPRA improved throughput from 16 min to 10 s per sample, and substrate peptides usage was reduced from 100 mM to 5 µM. When implementing the SPE-MS/MS-based DPRA into a high-throughput platform, we found 10 compounds that depleted lysine peptide and 24 compounds that depleted cysteine peptide (including 7 unreported chemicals from 55 compounds we tested) in a concentration-response manner. The adduct formation between cysteine and cinnamic aldehyde and ethylene glycol dimethacrylate were further analyzed using high-performance liquid chromatography time-of-flight mass spectrometry (HPLC-TOF-MS) to confirm the conjugation. Overall, the automated SPE-MS/MS-based platform is an efficient, economic, and accurate way to detect skin sensitizers.
Asunto(s)
Alérgenos/toxicidad , Cromatografía Líquida de Alta Presión , Dermatitis Alérgica por Contacto/etiología , Ensayos Analíticos de Alto Rendimiento , Péptidos/química , Piel/efectos de los fármacos , Espectrometría de Masas en Tándem , Pruebas de Toxicidad , Alérgenos/química , Alternativas a las Pruebas en Animales , Cromatografía Líquida de Alta Presión/normas , Cisteína , Ensayos Analíticos de Alto Rendimiento/normas , Humanos , Lisina , Estándares de Referencia , Reproducibilidad de los Resultados , Medición de Riesgo , Espectrometría de Masas en Tándem/normasRESUMEN
Quality control monitoring of cell lines utilized in biomedical research is of utmost importance and is critical for the reproducibility of data. Two key pitfalls in tissue culture are 1) cell line authenticity and 2) Mycoplasma contamination. As a collaborative research institute, the National Center for Advancing Translational Sciences (NCATS) receives cell lines from a range of commercial and academic sources, which are adapted for high-throughput screening. Here, we describe the implementation of routine NCATS-wide Mycoplasma testing and short tandem repeat (STR) testing for cell lines. Initial testing identified a >10% Mycoplasma contamination rate. While the implementation of systematic testing has not fully suppressed Mycoplasma contamination rates, clearly defined protocols that include the immediate destruction of contaminated cell lines wherever possible has enabled rapid intervention and removal of compromised cell lines. Data for >2000 cell line samples tested over 3 years, and case studies are provided. STR testing of 186 cell lines with established STR profiles revealed only five misidentified cell lines, all of which were received from external labs. The data collected over the 3 years since implementation of this systematic testing demonstrate the importance of continual vigilance for rapid identification of "problem" cell lines, for ensuring reproducible data in translational science research.
Asunto(s)
Técnicas de Cultivo de Célula/métodos , Mycoplasma/aislamiento & purificación , Control de Calidad , Investigación Biomédica Traslacional/normas , Línea Celular Tumoral , Humanos , Repeticiones de Microsatélite/genética , Mycoplasma/patogenicidad , National Center for Advancing Translational Sciences (U.S.) , Reacción en Cadena de la Polimerasa , Investigación Biomédica Traslacional/tendencias , Estados Unidos/epidemiologíaRESUMEN
Amyloid-ß (Aß) is best known as the misfolded peptide that is involved in the pathogenesis of Alzheimer's disease (AD), and it is currently the primary therapeutic target in attempts to arrest the course of this disease. This notoriety has overshadowed evidence that Aß serves several important physiological functions. Aß is present throughout the lifespan, it has been found in all vertebrates examined thus far, and its molecular sequence shows a high degree of conservation. These features are typical of a factor that contributes significantly to biological fitness, and this suggestion has been supported by evidence of functions that are beneficial for the brain. The putative roles of Aß include protecting the body from infections, repairing leaks in the blood-brain barrier, promoting recovery from injury, and regulating synaptic function. Evidence for these beneficial roles comes from in vitro and in vivo studies, which have shown that the cellular production of Aß rapidly increases in response to a physiological challenge and often diminishes upon recovery. These roles are further supported by the adverse outcomes of clinical trials that have attempted to deplete Aß in order to treat AD. We suggest that anti-Aß therapies will produce fewer adverse effects if the known triggers of Aß deposition (e.g., pathogens, hypertension, and diabetes) are addressed first.
RESUMEN
The amyloid-ß (Aß) peptide has long been considered to be the driving force behind Alzheimer's disease (AD). However, clinical trials that have successfully reduced Aß burden in the brain have not slowed the cognitive decline, and in some instances, have resulted in adverse outcomes. While these results can be interpreted in different ways, a more nuanced picture of Aß is emerging that takes into account the facts that the peptide is evolutionarily conserved and is present throughout life in cognitively normal individuals. Recent evidence indicates a role for Aß as an antimicrobial peptide (AMP), a class of innate immune defense molecule that utilizes fibrillation to protect the host from a wide range of infectious agents. In humans and in animal models, infection of the brain frequently leads to increased amyloidogenic processing of the amyloid-ß protein precursor (AßPP) and resultant fibrillary aggregates of Aß. Evidence from in vitro and in vivo studies demonstrates that Aß oligomers have potent, broad-spectrum antimicrobial properties by forming fibrils that entrap pathogens and disrupt cell membranes. Importantly, overexpression of Aß confers increased resistance to infection from both bacteria and viruses. The antimicrobial role of Aß may explain why increased rates of infection have been observed in some of the AD clinical trials that depleted Aß. Perhaps progress toward a cure for AD will accelerate once treatment strategies begin to take into account the likely physiological functions of this enigmatic peptide.