RESUMEN
Proteins and RNA functionally and physically intersect in multiple biological processes, however, currently no universal method is available to purify protein-RNA complexes. Here, we introduce XRNAX, a method for the generic purification of protein-crosslinked RNA, and demonstrate its versatility to study the composition and dynamics of protein-RNA interactions by various transcriptomic and proteomic approaches. We show that XRNAX captures all RNA biotypes and use this to characterize the sub-proteomes that interact with coding and non-coding RNAs (ncRNAs) and to identify hundreds of protein-RNA interfaces. Exploiting the quantitative nature of XRNAX, we observe drastic remodeling of the RNA-bound proteome during arsenite-induced stress, distinct from autophagy-related changes in the total proteome. In addition, we combine XRNAX with crosslinking immunoprecipitation sequencing (CLIP-seq) to validate the interaction of ncRNA with lamin B1 and EXOSC2. Thus, XRNAX is a resourceful approach to study structural and compositional aspects of protein-RNA interactions to address fundamental questions in RNA-biology.
Asunto(s)
Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Proteínas de Unión al ARN/aislamiento & purificación , ARN/aislamiento & purificación , Sitios de Unión , Complejo Multienzimático de Ribonucleasas del Exosoma/metabolismo , Humanos , Inmunoprecipitación/métodos , Lamina Tipo B/metabolismo , Unión Proteica/genética , Unión Proteica/fisiología , Biosíntesis de Proteínas/genética , Biosíntesis de Proteínas/fisiología , Procesamiento Proteico-Postraduccional , Proteínas/aislamiento & purificación , Proteínas/metabolismo , Proteoma/metabolismo , Proteómica/métodos , ARN/genética , ARN/metabolismo , ARN Mensajero/metabolismo , ARN no Traducido/metabolismo , Proteínas de Unión al ARN/metabolismo , TranscriptomaRESUMEN
Transcriptome profiling is widely used to infer functional states of specific cell types, as well as their responses to stimuli, to define contributions to physiology and pathophysiology. Focusing on microglia, the brain's macrophages, we report here a side-by-side comparison of classical cell-sorting-based transcriptome sequencing and the 'RiboTag' method, which avoids cell retrieval from tissue context and yields translatome sequencing information. Conventional whole-cell microglial transcriptomes were found to be significantly tainted by artifacts introduced by tissue dissociation, cargo contamination and transcripts sequestered from ribosomes. Conversely, our data highlight the added value of RiboTag profiling for assessing the lineage accuracy of Cre recombinase expression in transgenic mice. Collectively, this study indicates method-based biases, reveals observer effects and establishes RiboTag-based translatome profiling as a valuable complement to standard sorting-based profiling strategies.
Asunto(s)
Microglía , ARN Mensajero/análisis , Análisis de Secuencia de ARN/métodos , Animales , Inmunoprecipitación/métodos , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , RibosomasRESUMEN
LIN28 plays a critical role in developmental transition, glucose metabolism, and tumorigenesis. At the molecular level, LIN28 is known to repress maturation of let-7 microRNAs and enhance translation of certain mRNAs. In this study, we obtain a genome-wide view of the molecular function of LIN28A in mouse embryonic stem cells by carrying out RNA crosslinking-immunoprecipitation-sequencing (CLIP-seq) and ribosome footprinting. We find that, in addition to let-7 precursors, LIN28A binds to a large number of spliced mRNAs. LIN28A recognizes AAGNNG, AAGNG, and less frequently UGUG, which are located in the terminal loop of a small hairpin. LIN28A is localized to the periendoplasmic reticulum (ER) area and inhibits translation of mRNAs that are destined for the ER, reducing the synthesis of transmembrane proteins, ER or Golgi lumen proteins, and secretory proteins. Our study suggests a selective regulatory mechanism for ER-associated translation and reveals an unexpected role of LIN28A as a global suppressor of genes in the secretory pathway.
Asunto(s)
Biosíntesis de Proteínas , ARN Mensajero/metabolismo , Proteínas de Unión al ARN/metabolismo , Animales , Células Madre Embrionarias/metabolismo , Secuenciación de Nucleótidos de Alto Rendimiento , Inmunoprecipitación/métodos , Ratones , MicroARNs/metabolismo , Ribosomas/metabolismo , Vías Secretoras , Análisis de Secuencia de ARNRESUMEN
RNA-binding proteins (RBPs) are essential for RNA metabolism and profoundly impact health and disease. The subcellular organization of RBP interaction networks with target RNAs remains largely unexplored. Here, we develop colocalization CLIP (coCLIP), a method that combines cross-linking and immunoprecipitation (CLIP) with proximity labeling, to explore in-depth the subcellular RNA interactions of the RBP human antigen R (HuR). Using this method, we uncover HuR's dynamic and location-specific interactions with RNA, revealing alterations in sequence preferences and interactions in the nucleus, cytosol, or stress granule (SG) compartments. We uncover HuR's unique binding preferences within SGs during arsenite stress, illuminating intricate interactions that conventional methodologies cannot capture. Overall, coCLIP provides a powerful method for revealing RBP-RNA interactions based on localization and lays the foundation for an advanced understanding of RBP models that incorporate subcellular location as a critical determinant of their functions.
Asunto(s)
Unión Proteica , Proteínas de Unión al ARN , ARN , Humanos , ARN/metabolismo , ARN/genética , Proteínas de Unión al ARN/metabolismo , Proteínas de Unión al ARN/genética , Inmunoprecipitación/métodos , Proteína 1 Similar a ELAV/metabolismo , Proteína 1 Similar a ELAV/genética , Núcleo Celular/metabolismo , Núcleo Celular/genética , Gránulos Citoplasmáticos/metabolismo , Arsenitos , Células HeLa , Citosol/metabolismo , Células HEK293RESUMEN
RNA binding proteins (RBPs) regulate all aspects in the life cycle of RNA molecules. To elucidate the elements that guide RNA specificity, regulatory mechanisms, and functions of RBPs, methods that identify direct endogenous protein-RNA interactions are particularly valuable. UV crosslinking and immunoprecipitation (CLIP) purifies short RNA fragments that crosslink to a specific protein and then identifies these fragments by sequencing. When combined with high-throughput sequencing, CLIP can produce transcriptome-wide maps of RNA crosslink sites. The protocol is comprised of several dozen biochemical steps, and improvements made over the last 15 years have increased its resolution, sensitivity, and convenience. Adaptations of CLIP are also emerging in the epitranscriptomic field to map the positions of RNA modifications accurately. Here, we describe the rationale for each step in the protocol and discuss the impact of variations to help users determine the most suitable option.
Asunto(s)
Inmunoprecipitación/métodos , Proteínas con Motivos de Reconocimiento de ARN/genética , ARN/genética , Sitios de Unión , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Unión Proteica , Proteínas de Unión al ARN/metabolismo , Análisis de Secuencia de ARN/métodos , TranscriptomaRESUMEN
Compared to noncoding RNAs (ncRNAs), such as rRNAs and ribozymes, for which high-resolution structures abound, little is known about the tertiary structures of mRNAs. In eukaryotic cells, newly made mRNAs are packaged with proteins in highly compacted mRNA particles (mRNPs), but the manner of this mRNA compaction is unknown. Here, we developed and implemented RIPPLiT (RNA immunoprecipitation and proximity ligation in tandem), a transcriptome-wide method for probing the 3D conformations of RNAs stably associated with defined proteins, in this case, exon junction complex (EJC) core factors. EJCs multimerize with other mRNP components to form megadalton-sized complexes that protect large swaths of newly synthesized mRNAs from endonuclease digestion. Unlike ncRNPs, wherein strong locus-specific structures predominate, mRNPs behave more like flexible polymers. Polymer analysis of proximity ligation data for hundreds of mRNA species demonstrates that nascent and pre-translational mammalian mRNAs are compacted by their associated proteins into linear rod-like structures.
Asunto(s)
Precursores del ARN/ultraestructura , Ribonucleoproteínas/genética , Ribonucleoproteínas/ultraestructura , Núcleo Celular , Exones , Células HEK293 , Humanos , Inmunoprecipitación/métodos , Procesamiento Proteico-Postraduccional , Precursores del ARN/genética , Empalme del ARN , Estabilidad del ARN , ARN Mensajero/genética , ARN Mensajero/ultraestructura , ARN no Traducido , Empalmosomas , Transcripción GenéticaRESUMEN
Protein kinase RNA-activated (PKR) induces immune response by sensing viral double-stranded RNAs (dsRNAs). However, growing evidence suggests that PKR can also be activated by endogenously expressed dsRNAs. Here, we capture these dsRNAs by formaldehyde-mediated crosslinking and immunoprecipitation sequencing and find that various noncoding RNAs interact with PKR. Surprisingly, the majority of the PKR-interacting RNA repertoire is occupied by mitochondrial RNAs (mtRNAs). MtRNAs can form intermolecular dsRNAs owing to bidirectional transcription of the mitochondrial genome and regulate PKR and eIF2α phosphorylation to control cell signaling and translation. Moreover, PKR activation by mtRNAs is counteracted by PKR phosphatases, disruption of which causes apoptosis from PKR overactivation even in uninfected cells. Our work unveils dynamic regulation of PKR even without infection and establishes PKR as a sensor for nuclear and mitochondrial signaling cues in regulating cellular metabolism.
Asunto(s)
eIF-2 Quinasa/metabolismo , eIF-2 Quinasa/fisiología , Línea Celular , Núcleo Celular , Activación Enzimática , Factor 2 Eucariótico de Iniciación/metabolismo , Células HEK293 , Células HeLa , Humanos , Inmunoprecipitación/métodos , Mitocondrias/genética , Fosforilación , ARN Bicatenario/genética , ARN Mitocondrial/genética , ARN Mitocondrial/fisiología , ARN no Traducido/genética , ARN no Traducido/fisiología , Transducción de Señal , eIF-2 Quinasa/inmunologíaRESUMEN
MicroRNA-122, an abundant and conserved liver-specific miRNA, regulates hepatic metabolism and functions as a tumor suppressor, yet systematic and direct biochemical elucidation of the miR-122 target network remains incomplete. To this end, we performed Argonaute crosslinking immunoprecipitation (Argonaute [Ago]-CLIP) sequencing in miR-122 knockout and control mouse livers, as well as in matched human hepatocellular carcinoma (HCC) and benign liver tissue to identify miRNA target sites transcriptome-wide in two species. We observed a majority of miR-122 binding on 3' UTRs and coding exons followed by extensive binding to other genic and non-genic sites. Motif analysis of miR-122-dependent binding revealed a G-bulged motif in addition to canonical motifs. A large number of miR-122 targets were found to be species specific. Upregulation of several common mouse and human targets, most notably BCL9, predicted survival in HCC patients. These results broadly define the molecular consequences of miR-122 downregulation in hepatocellular carcinoma.
Asunto(s)
Proteínas Argonautas/genética , Carcinoma Hepatocelular/genética , Inmunoprecipitación/métodos , Neoplasias Hepáticas/genética , MicroARNs/genética , Transcriptoma , Regiones no Traducidas 3' , Animales , Proteínas Argonautas/metabolismo , Sitios de Unión , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/mortalidad , Carcinoma Hepatocelular/patología , Estudios de Casos y Controles , Línea Celular Tumoral , Proliferación Celular , Biología Computacional , Bases de Datos Genéticas , Regulación hacia Abajo , Exones , Regulación Neoplásica de la Expresión Génica , Estudio de Asociación del Genoma Completo , Genotipo , Humanos , Péptidos y Proteínas de Señalización Intracelular/genética , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/mortalidad , Neoplasias Hepáticas/patología , Ratones , Ratones Noqueados , MicroARNs/metabolismo , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Fenotipo , Interferencia de ARN , Especificidad de la Especie , Factores de Tiempo , Factores de Transcripción , Transcripción Genética , Transfección , Vía de Señalización WntRESUMEN
Intact-mass spectrometry has huge potential for clinical application, as it enables both quantitative and qualitative analysis of intact proteins and possibly unlocks additional pathophysiological information via, e.g., detection of specific post-translational modifications (PTMs). Such valuable and clinically useful selectivity is typically lost during conventional bottom-up mass spectrometry. We demonstrate an innovative immunoprecipitation protein enrichment assay coupled to ultrahigh performance liquid chromatography quadrupole time-of-flight high resolution mass spectrometry (UPLC-QToF-HRMS) for the fast and simple identification of the protein tumor marker Neuron Specific Enolase Gamma (NSEγ) at low endogenous concentrations in human serum. Additionally, using the combination of immunoaffinity purification with intact mass spectrometry, the presence of NSEγ in an acetylated form in human serum was detected. This highlights the unique potential of immunoaffinity intact mass spectrometry in clinical diagnostics.
Asunto(s)
Biomarcadores de Tumor , Espectrometría de Masas , Fosfopiruvato Hidratasa , Fosfopiruvato Hidratasa/sangre , Fosfopiruvato Hidratasa/aislamiento & purificación , Humanos , Biomarcadores de Tumor/sangre , Biomarcadores de Tumor/metabolismo , Acetilación , Espectrometría de Masas/métodos , Procesamiento Proteico-Postraduccional , Inmunoprecipitación/métodos , Cromatografía Líquida de Alta Presión/métodosRESUMEN
Immunoprecipitation (IP) and co-immunoprecipitation (co-IP) are well-established methodologies to analyze protein expression and intermolecular interaction. Composition of extraction and washing buffer for preparing protein is important to accomplish experimental purpose. Various kinds of detergents are included in buffer to adjust extraction efficiency and washing effect. Among them, Triton X-100 (Tx-100), Nonidet P-40 (NP40), deoxycholic acid (DOC) and SDS are generally used according to experimental purpose and characteristic features of protein of interest. In some cases, general detergents disrupt intermolecular interaction and make it impossible to analyze molecular relation of protein of interest with its binding partners. In this study, we propose saponin, a natural detergent, is useful for co-immunoprecipitation when analyzing fragile intermolecular interactions, in which dystrophin and dystroglycan are used as a representative interaction. One of the most notable findings in this report is that intermolecular association between dystrophin and dystroglycan is maintained in saponin buffer whereas general detergents, such as Tx-100, NP40 and DOC, dissociate its binding. Furthermore, supplementation of trehalose, which has been shown to act as a molecular chaperone, facilitates efficient detection of dystrophin-dystroglycan macromolecular complex in co-IP assay. Importantly, the extraction buffer comprising 3 % saponin, 0.5 M trehalose and 0.05 % Tx-100 (we named it STX buffer) is applicable to co-IP for another molecular interaction, N-cadherin and ß-catenin, indicating that this methodology can be used for versatile proteins of interest. Thus, STX buffer emerges as an alternative extraction method useful for analyzing fragile intermolecular associations and provides opportunity to identify complex interactomes, which may facilitate proteome-research and functional analysis of proteins of interest.
Asunto(s)
Saponinas , Trehalosa , Saponinas/química , Trehalosa/química , Inmunoprecipitación/métodos , Animales , Detergentes/química , Unión Proteica , Humanos , Octoxinol/químicaRESUMEN
STAU2 is a double-stranded RNA-binding protein enriched in the nervous system. During asymmetric divisions in the developing mouse cortex, STAU2 preferentially distributes into the intermediate progenitor cell (IPC), delivering RNA molecules that can impact IPC behavior. Corticogenesis occurs on a precise time schedule, raising the hypothesis that the cargo STAU2 delivers into IPCs changes over time. To test this, we combine RNA-immunoprecipitation with sequencing (RIP-seq) over four stages of mouse cortical development, generating a comprehensive cargo profile for STAU2. A subset of the cargo was 'stable', present at all stages, and involved in chromosome organization, macromolecule localization, translation and DNA repair. Another subset was 'dynamic', changing with cortical stage, and involved in neurogenesis, cell projection organization, neurite outgrowth, and included cortical layer markers. Notably, the dynamic STAU2 cargo included determinants of IPC versus neuronal fates and genes contributing to abnormal corticogenesis. Knockdown of one STAU2 target, Taf13, previously linked to microcephaly and impaired myelination, reduced oligodendrogenesis in vitro. We conclude that STAU2 contributes to the timing of corticogenesis by binding and delivering complex and temporally regulated RNA cargo into IPCs.
Asunto(s)
Corteza Cerebral/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Proteínas de Unión al ARN/metabolismo , ARN/metabolismo , Células Madre/metabolismo , Animales , Células Cultivadas , Reparación del ADN/fisiología , Femenino , Inmunoprecipitación/métodos , Masculino , Ratones , Neurogénesis/fisiología , Neuronas/metabolismo , EmbarazoRESUMEN
The RNA field has been revolutionized by methods that allow genome-scale identification of RNA-protein interaction sites. Two reports now introduce more efficient approaches, opening the technology to wider adoption (Van Nostrand et al., 2016; Zarnegar et al., 2016).
Asunto(s)
Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Inmunoprecipitación/métodos , Proteínas de Unión al ARN/metabolismo , ARN/metabolismo , Análisis de Secuencia de ADN/métodos , Análisis de Secuencia de Proteína/métodos , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Humanos , Motivos de Nucleótidos , Unión Proteica , ARN/química , ARN/genética , Motivos de Unión al ARN , Proteínas de Unión al ARN/química , Proteínas de Unión al ARN/genéticaRESUMEN
To identify endogenous miRNA-target sites, we isolated AGO-bound RNAs from Caenorhabditis elegans by individual-nucleotide resolution crosslinking immunoprecipitation (iCLIP), which fortuitously also produced miRNA-target chimeric reads. Through the analysis of thousands of reproducible chimeras, pairing to the miRNA seed emerged as the predominant motif associated with functional interactions. Unexpectedly, we discovered that additional pairing to 3' sequences is prevalent in the majority of target sites and leads to specific targeting by members of miRNA families. By editing an endogenous target site, we demonstrate that 3' pairing determines targeting by specific miRNA family members and that seed pairing is not always sufficient for functional target interactions. Finally, we present a simplified method, chimera PCR (ChimP), for the detection of specific miRNA-target interactions. Overall, our analysis revealed that sequences in the 5' as well as the 3' regions of a miRNA provide the information necessary for stable and specific miRNA-target interactions in vivo.
Asunto(s)
Proteínas de Caenorhabditis elegans/genética , Caenorhabditis elegans/genética , MicroARNs/genética , ARN de Helminto/genética , ARN Mensajero/genética , Proteínas de Unión al ARN/genética , Animales , Emparejamiento Base , Secuencia de Bases , Sitios de Unión , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/metabolismo , Exones , Regulación de la Expresión Génica , Inmunoprecipitación/métodos , Intrones , MicroARNs/clasificación , MicroARNs/metabolismo , Unión Proteica , ARN de Helminto/metabolismo , ARN Largo no Codificante/genética , ARN Largo no Codificante/metabolismo , ARN Mensajero/metabolismo , ARN Nucleolar Pequeño/genética , ARN Nucleolar Pequeño/metabolismo , Proteínas de Unión al ARN/metabolismoRESUMEN
Timely and accurate diagnosis of Schistosoma infection is important to adopt effective strategies for schistosomiasis control. Previously, we demonstrated that Schistosoma japonicum can secret extracellular vesicles and their cargos may serve as a novel type of biomarkers for diagnosing schistosomiasis. Here, we developed a Gaussia luciferase immunoprecipitation assay combined with S. japonicum extracellular vesicle (SjEV) protein to evaluate its potential for diagnosing schistosomiasis. A saposin-like protein (SjSLP) identified from SjEVs was fused to the Gaussia luciferase as the diagnostic antigen. The developed method showed good capability for detecting S. japonicum infection in mice and human patients. We also observed that the method could detect Schistosoma infection in mice as early as 7 days of post-infection, which showed better sensitivity than that of indirect ELISA method. Overall, the developed method showed a good potential for detecting Schistosoma infection particularly for early stage, which may provide an alternative strategy for identify Schistosoma infection for disease control.
Asunto(s)
Inmunoprecipitación , Luciferasas , Schistosoma japonicum , Esquistosomiasis Japónica , Animales , Esquistosomiasis Japónica/diagnóstico , Esquistosomiasis Japónica/parasitología , Schistosoma japonicum/enzimología , Schistosoma japonicum/inmunología , Ratones , Humanos , Inmunoprecipitación/métodos , Luciferasas/genética , Femenino , Sensibilidad y Especificidad , Ratones Endogámicos BALB C , Ensayo de Inmunoadsorción Enzimática/métodos , Vesículas Extracelulares , Antígenos Helmínticos/análisis , Antígenos Helmínticos/inmunología , MasculinoRESUMEN
There have been many attempts in pharmaceutical industries and academia to improve the pharmacokinetic characteristics of anti-tumor small-molecule drugs by conjugating them with large molecules, such as monoclonal antibodies, called ADCs. In this context, albumin, one of the most abundant proteins in the blood, has also been proposed as a large molecule to be conjugated with anti-cancer small-molecule drugs. The half-life of albumin is 3 weeks in humans, and its distribution to tumors is higher than in normal tissues. However, few studies have been conducted for the in vivo prepared albumin-drug conjugates, possibly due to the lack of robust bioanalytical methods, which are critical for evaluating the ADME/PK properties of in vivo prepared albumin-drug conjugates. In this study, we developed a bioanalytical method of the albumin-conjugated MAC glucuronide phenol linked SN-38 ((2S,3S,4S,5R,6S)-6-(4-(((((((S)-4,11-diethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano [3',4':6,7] indolizino [1,2-b] quinolin-9-yl)oxy)methyl)(2 (methylsulfonyl)ethyl)carbamoyl)oxy)methyl)-2-(2-(3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-N-methylpropanamido)acetamido)phenoxy)-3,4,5-trihydroxytetra-hydro-2H-pyran-2-carboxylic acid) as a proof-of-concept. This method is based on immunoprecipitation using magnetic beads and the quantification of albumin-conjugated drug concentration using LC-qTOF/MS in mouse plasma. Finally, the developed method was applied to the in vivo intravenous (IV) mouse pharmacokinetic study of MAC glucuronide phenol-linked SN-38.
Asunto(s)
Albúminas , Inmunoprecipitación , Irinotecán , Cromatografía Líquida con Espectrometría de Masas , Animales , Humanos , Ratones , Albúminas/química , Albúminas/farmacocinética , Glucuronidasa/metabolismo , Glucurónidos/química , Glucurónidos/metabolismo , Inmunoprecipitación/métodos , Irinotecán/sangre , Irinotecán/química , Irinotecán/metabolismo , Irinotecán/farmacocinética , Cromatografía Líquida con Espectrometría de Masas/métodos , Magnetismo , Fenol/químicaRESUMEN
In eukaryotic cells, proteins that associate with RNA regulate its activity to control cellular function. To fully illuminate the basis of RNA function, it is essential to identify such RNA-associated proteins, their mode of action on RNA, and their preferred RNA targets and binding sites. By analyzing catalogs of human RNA-associated proteins defined by ultraviolet light (UV)-dependent and -independent approaches, we classify these proteins into two major groups: (i) the widely recognized RNA binding proteins (RBPs), which bind RNA directly and UV-crosslink efficiently to RNA, and (ii) a new group of RBP-associated factors (RAFs), which bind RNA indirectly via RBPs and UV-crosslink poorly to RNA. As the UV crosslinking and immunoprecipitation followed by sequencing (CLIP-seq) approach will be unsuitable to identify binding sites of RAFs, we show that formaldehyde crosslinking stabilizes RAFs within ribonucleoproteins to allow for their immunoprecipitation under stringent conditions. Using an RBP (CASC3) and an RAF (RNPS1) within the exon junction complex (EJC) as examples, we show that formaldehyde crosslinking combined with RNA immunoprecipitation in tandem followed by sequencing (xRIPiT-seq) far exceeds CLIP-seq to identify binding sites of RNPS1. xRIPiT-seq reveals that RNPS1 occupancy is increased on exons immediately upstream of strong recursively spliced exons, which depend on the EJC for their inclusion.
Asunto(s)
Sitios de Unión/genética , Unión Proteica/genética , ARN/química , ARN/genética , Ribonucleoproteínas/genética , Ribonucleoproteínas/metabolismo , Línea Celular , Células Eucariotas/metabolismo , Exones/genética , Células HEK293 , Humanos , Inmunoprecipitación/métodos , Empalme del ARN/genética , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismo , Transcriptoma/genéticaRESUMEN
The inert chemical property of RNA modification N6-methyladenosine (m6A) makes it very challenging to detect. Most m6A sequencing methods rely on m6A-antibody immunoprecipitation and cannot distinguish m6A and N6,2'-O-dimethyladenosine modification at the cap +1 position (cap m6Am). Although the two antibody-free methods (m6A-REF-seq/MAZTER-seq and DART-seq) have been developed recently, they are dependent on m6A sequence or cellular transfection. Here, we present an antibody-free, FTO-assisted chemical labeling method termed m6A-SEAL for specific m6A detection. We applied m6A-SEAL to profile m6A landscapes in humans and plants, which displayed the known m6A distribution features in transcriptome. By doing a comparison with all available m6A sequencing methods and specific m6A sites validation by SELECT, we demonstrated that m6A-SEAL has good sensitivity, specificity and reliability for transcriptome-wide detection of m6A. Given its tagging ability and FTO's oxidation property, m6A-SEAL enables many applications such as enrichment, imaging and sequencing to drive future functional studies of m6A and other modifications.
Asunto(s)
Adenosina/análogos & derivados , Marcadores de Afinidad/química , Adenosina/análisis , Adenosina/química , Dioxigenasa FTO Dependiente de Alfa-Cetoglutarato/química , Dioxigenasa FTO Dependiente de Alfa-Cetoglutarato/metabolismo , Humanos , Inmunoprecipitación/métodos , Metilación , ARN/química , ARN Mensajero/química , ARN Mensajero/metabolismo , Reproducibilidad de los Resultados , Sensibilidad y Especificidad , TranscriptomaRESUMEN
Autophagy-related protein 8 (ATG8) is a highly conserved ubiquitin-like protein that modulates autophagy pathways by binding autophagic membranes and a number of proteins, including cargo receptors and core autophagy components. Throughout plant evolution, ATG8 has expanded from a single protein in algae to multiple isoforms in higher plants. However, the degree to which ATG8 isoforms have functionally specialized to bind distinct proteins remains unclear. Here, we describe a comprehensive protein-protein interaction resource, obtained using in planta immunoprecipitation (IP) followed by mass spectrometry (MS), to define the potato ATG8 interactome. We discovered that ATG8 isoforms bind distinct sets of plant proteins with varying degrees of overlap. This prompted us to define the biochemical basis of ATG8 specialization by comparing two potato ATG8 isoforms using both in vivo protein interaction assays and in vitro quantitative binding affinity analyses. These experiments revealed that the N-terminal ß-strand-and, in particular, a single amino acid polymorphism-underpins binding specificity to the substrate PexRD54 by shaping the hydrophobic pocket that accommodates this protein's ATG8-interacting motif (AIM). Additional proteomics experiments indicated that the N-terminal ß-strand shapes the broader ATG8 interactor profiles, defining interaction specificity with about 80 plant proteins. Our findings are consistent with the view that ATG8 isoforms comprise a layer of specificity in the regulation of selective autophagy pathways in plants.
Asunto(s)
Familia de las Proteínas 8 Relacionadas con la Autofagia/metabolismo , Autofagia , Proteínas de Plantas/metabolismo , Plantas/metabolismo , Familia de las Proteínas 8 Relacionadas con la Autofagia/química , Familia de las Proteínas 8 Relacionadas con la Autofagia/genética , Inmunoprecipitación/métodos , Espectrometría de Masas/métodos , Filogenia , Proteínas de Plantas/química , Proteínas de Plantas/genética , Plantas/clasificación , Plantas/genética , Plantas Modificadas Genéticamente , Unión Proteica , Conformación Proteica en Lámina beta , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Proteómica/métodos , Solanum tuberosum/genética , Solanum tuberosum/metabolismo , Nicotiana/genética , Nicotiana/metabolismoRESUMEN
RATIONALE: Doxorubicin is one of the most potent antitumor agents available; however, its clinical use is restricted because it poses a risk of severe cardiotoxicity. Previous work has established that CircITCH (circular RNA ITCH [E3 ubiquitin-protein ligase]) is a broad-spectrum tumor-suppressive circular RNA and that its host gene, ITCH (E3 ubiquitin protein ligase), is involved in doxorubicin-induced cardiotoxicity (DOXIC). Whether CircITCH plays a role in DOXIC remains unknown. OBJECTIVE: We aimed to dissect the role of CircITCH in DOXIC and further decipher its potential mechanisms. METHODS AND RESULTS: Circular RNA sequencing was performed to screen the potentially involved circRNAs in DOXI pathogenesis. Quantitative polymerase chain reaction and RNA in situ hybridization revealed that CircITCH was downregulated in doxorubicin-treated human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) as well as in the autopsy specimens from cancer patients who suffered from doxorubicin-induced cardiomyopathy. Cell death/viability assays, detection of cardiomyocyte necrosis markers, microelectrode array, and cardiomyocyte functional assays revealed that CircITCH ameliorated doxorubicin-induced cardiomyocyte injury and dysfunction. Detection of cellular/mitochondrial oxidative stress and DNA damage markers verified that CircITCH alleviated cellular/mitochondrial oxidative stress and DNA damage induced by doxorubicin. RNA pull-down assays, Ago2 immunoprecipitation and double fluorescent in situ hybridization identified miR-330-5p as a direct target of CircITCH. Moreover, CircITCH was found to function by acting as an endogenous sponge that sequestered miR-330-5p. Bioinformatic analysis, luciferase reporter assays, and quantitative polymerase chain reaction showed that SIRT6 (sirtuin 6), BIRC5 (baculoviral IAP repeat containing 5, Survivin), and ATP2A2 (ATPase sarcoplasmic/endoplasmic reticulum Ca2+ transporting 2, SERCA2a [SR Ca2+-ATPase 2]) were direct targets of miR-330-5p and that they were regulated by the CircITCH/miR-330-5p axis in DOXIC. Further experiments demonstrated that CircITCH-mediated alleviation of DOXIC was dependent on the interactions between miR-330-5p and the 3'-UTRs of SIRT6, BIRC5, and ATP2A2 mRNA. Finally, AAV9 (adeno-associated virus serotype 9) vector-based overexpression of the well-conserved CircITCH partly prevented DOXIC in mice. CONCLUSIONS: CircITCH represents a novel therapeutic target for DOXIC because it acts as a natural sponge of miR-330-5p, thereby upregulating SIRT6, Survivin and SERCA2a to alleviate doxorubicin-induced cardiomyocyte injury and dysfunction.
Asunto(s)
Antibióticos Antineoplásicos/efectos adversos , Doxorrubicina/efectos adversos , MicroARNs/metabolismo , ARN Circular/fisiología , Proteínas Represoras/metabolismo , ATPasas Transportadoras de Calcio del Retículo Sarcoplásmico/metabolismo , Sirtuinas/metabolismo , Survivin/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Regiones no Traducidas 3'/genética , Adenovirus Humanos , Animales , Proteínas Argonautas/análisis , Sitios de Unión , Biomarcadores , Cardiotoxicidad/genética , Cardiotoxicidad/metabolismo , Cardiotoxicidad/terapia , Muerte Celular , Supervivencia Celular , Daño del ADN , Regulación hacia Abajo , Silenciador del Gen , Genes Supresores de Tumor , Humanos , Inmunoprecipitación/métodos , Hibridación Fluorescente in Situ/métodos , Ratones , MicroARNs/genética , Mitocondrias Cardíacas/metabolismo , Mutación , Contracción Miocárdica/fisiología , Miocitos Cardíacos/efectos de los fármacos , Miocitos Cardíacos/metabolismo , Miocitos Cardíacos/patología , Necrosis , Estrés Oxidativo , ARN Circular/efectos de los fármacos , Proteínas Represoras/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , ATPasas Transportadoras de Calcio del Retículo Sarcoplásmico/genética , Survivin/genética , Ubiquitina-Proteína Ligasas/genética , Regulación hacia ArribaRESUMEN
Mutations that increase the protein kinase activity of LRRK2 are one of the most common causes of familial Parkinson's disease. LRRK2 phosphorylates a subset of Rab GTPases within their Switch-II motif, impacting interaction with effectors. We describe and validate a new, multiplexed targeted mass spectrometry assay to quantify endogenous levels of LRRK2-phosphorylated Rab substrates (Rab1, Rab3, Rab8, Rab10, Rab35 and Rab43) as well as total levels of Rabs, LRRK2 and LRRK2-phosphorylated at the Ser910 and Ser935 biomarker sites. Exploiting this assay, we quantify for the first time the relative levels of each of the pRab proteins in different cells (mouse embryonic fibroblasts, human neutrophils) and mouse tissues (brain, kidney, lung and spleen). We define how these components are impacted by Parkinson's pathogenic mutations (LRRK2[R1441C] and VPS35[D620N]) and LRRK2 inhibitors. We find that the VPS35[D620N], but not LRRK2[R1441C] mutation, enhances Rab1 phosphorylation in a manner blocked by administration of an LRRK2 inhibitor, providing the first evidence that endogenous Rab1 is a physiological substrate for LRRK2. We exploit this assay to demonstrate that in Parkinson's patients with VPS35[D620N] mutations, phosphorylation of multiple Rab proteins (Rab1, Rab3, Rab8, Rab10 and Rab43) is elevated. We highlight the benefits of this assay over immunoblotting approaches currently deployed to assess LRRK2 Rab signalling pathway.