RESUMEN
Cell cycle progression relies on coordinated changes in the composition and subcellular localization of the proteome. By applying two distinct convolutional neural networks on images of millions of live yeast cells, we resolved proteome-level dynamics in both concentration and localization during the cell cycle, with resolution of â¼20 subcellular localization classes. We show that a quarter of the proteome displays cell cycle periodicity, with proteins tending to be controlled either at the level of localization or concentration, but not both. Distinct levels of protein regulation are preferentially utilized for different aspects of the cell cycle, with changes in protein concentration being mostly involved in cell cycle control and changes in protein localization in the biophysical implementation of the cell cycle program. We present a resource for exploring global proteome dynamics during the cell cycle, which will aid in understanding a fundamental biological process at a systems level.
Asunto(s)
Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Células Eucariotas/metabolismo , Redes Neurales de la Computación , Proteoma/metabolismo , Saccharomyces cerevisiae/citología , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMEN
The molecular pathology of multi-organ injuries in COVID-19 patients remains unclear, preventing effective therapeutics development. Here, we report a proteomic analysis of 144 autopsy samples from seven organs in 19 COVID-19 patients. We quantified 11,394 proteins in these samples, in which 5,336 were perturbed in the COVID-19 patients compared to controls. Our data showed that cathepsin L1, rather than ACE2, was significantly upregulated in the lung from the COVID-19 patients. Systemic hyperinflammation and dysregulation of glucose and fatty acid metabolism were detected in multiple organs. We also observed dysregulation of key factors involved in hypoxia, angiogenesis, blood coagulation, and fibrosis in multiple organs from the COVID-19 patients. Evidence for testicular injuries includes reduced Leydig cells, suppressed cholesterol biosynthesis, and sperm mobility. In summary, this study depicts a multi-organ proteomic landscape of COVID-19 autopsies that furthers our understanding of the biological basis of COVID-19 pathology.
Asunto(s)
COVID-19/metabolismo , Regulación de la Expresión Génica , Proteoma/biosíntesis , Proteómica , SARS-CoV-2/metabolismo , Autopsia , COVID-19/patología , COVID-19/terapia , Femenino , Humanos , Masculino , Especificidad de ÓrganosRESUMEN
Components of the proteostasis network malfunction in aging, and reduced protein quality control in neurons has been proposed to promote neurodegeneration. Here, we investigate the role of chaperone-mediated autophagy (CMA), a selective autophagy shown to degrade neurodegeneration-related proteins, in neuronal proteostasis. Using mouse models with systemic and neuronal-specific CMA blockage, we demonstrate that loss of neuronal CMA leads to altered neuronal function, selective changes in the neuronal metastable proteome, and proteotoxicity, all reminiscent of brain aging. Imposing CMA loss on a mouse model of Alzheimer's disease (AD) has synergistic negative effects on the proteome at risk of aggregation, thus increasing neuronal disease vulnerability and accelerating disease progression. Conversely, chemical enhancement of CMA ameliorates pathology in two different AD experimental mouse models. We conclude that functional CMA is essential for neuronal proteostasis through the maintenance of a subset of the proteome with a higher risk of misfolding than the general proteome.
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Envejecimiento/metabolismo , Enfermedad de Alzheimer/metabolismo , Encéfalo/metabolismo , Autofagia Mediada por Chaperones/fisiología , Neuronas/metabolismo , Proteostasis , Envejecimiento/patología , Enfermedad de Alzheimer/patología , Animales , Encéfalo/patología , Quinasa de la Caseína I/genética , Autofagia Mediada por Chaperones/genética , Modelos Animales de Enfermedad , Femenino , Masculino , Ratones , Neuronas/patología , ProteomaRESUMEN
Quantitative mass spectrometry has established proteome-wide regulation of protein abundance and post-translational modifications in various biological processes. Here, we used quantitative mass spectrometry to systematically analyze the thermal stability and solubility of proteins on a proteome-wide scale during the eukaryotic cell cycle. We demonstrate pervasive variation of these biophysical parameters with most changes occurring in mitosis and G1. Various cellular pathways and components vary in thermal stability, such as cell-cycle factors, polymerases, and chromatin remodelers. We demonstrate that protein thermal stability serves as a proxy for enzyme activity, DNA binding, and complex formation in situ. Strikingly, a large cohort of intrinsically disordered and mitotically phosphorylated proteins is stabilized and solubilized in mitosis, suggesting a fundamental remodeling of the biophysical environment of the mitotic cell. Our data represent a rich resource for cell, structural, and systems biologists interested in proteome regulation during biological transitions.
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Ciclo Celular , ADN/análisis , Proteoma/análisis , Proteómica/métodos , Ensamble y Desensamble de Cromatina , Análisis por Conglomerados , Células HeLa , Calor , Humanos , Espectrometría de Masas , Mitosis , Fosforilación , Procesamiento Proteico-Postraduccional , Estabilidad Proteica , ARN Polimerasa II/metabolismo , SolubilidadRESUMEN
We conducted comprehensive integrative molecular analyses of the complete set of tumors in The Cancer Genome Atlas (TCGA), consisting of approximately 10,000 specimens and representing 33 types of cancer. We performed molecular clustering using data on chromosome-arm-level aneuploidy, DNA hypermethylation, mRNA, and miRNA expression levels and reverse-phase protein arrays, of which all, except for aneuploidy, revealed clustering primarily organized by histology, tissue type, or anatomic origin. The influence of cell type was evident in DNA-methylation-based clustering, even after excluding sites with known preexisting tissue-type-specific methylation. Integrative clustering further emphasized the dominant role of cell-of-origin patterns. Molecular similarities among histologically or anatomically related cancer types provide a basis for focused pan-cancer analyses, such as pan-gastrointestinal, pan-gynecological, pan-kidney, and pan-squamous cancers, and those related by stemness features, which in turn may inform strategies for future therapeutic development.
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Neoplasias/patología , Aneuploidia , Cromosomas/genética , Análisis por Conglomerados , Islas de CpG , Metilación de ADN , Bases de Datos Factuales , Humanos , MicroARNs/metabolismo , Mutación , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Neoplasias/genética , ARN Mensajero/metabolismoRESUMEN
Although host responses to the ancestral SARS-CoV-2 strain are well described, those to the new Omicron variants are less resolved. We profiled the clinical phenomes, transcriptomes, proteomes, metabolomes, and immune repertoires of >1,000 blood cell or plasma specimens from SARS-CoV-2 Omicron patients. Using in-depth integrated multi-omics, we dissected the host response dynamics during multiple disease phases to reveal the molecular and cellular landscapes in the blood. Specifically, we detected enhanced interferon-mediated antiviral signatures of platelets in Omicron-infected patients, and platelets preferentially formed widespread aggregates with leukocytes to modulate immune cell functions. In addition, patients who were re-tested positive for viral RNA showed marked reductions in B cell receptor clones, antibody generation, and neutralizing capacity against Omicron. Finally, we developed a machine learning model that accurately predicted the probability of re-positivity in Omicron patients. Our study may inspire a paradigm shift in studying systemic diseases and emerging public health concerns.
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Plaquetas , COVID-19 , Humanos , SARS-CoV-2 , Infección Irruptiva , Multiómica , Anticuerpos Neutralizantes , Anticuerpos AntiviralesRESUMEN
RNA editing, a post-transcriptional process, allows the diversification of proteomes beyond the genomic blueprint; however it is infrequently used among animals for this purpose. Recent reports suggesting increased levels of RNA editing in squids thus raise the question of the nature and effects of these events. We here show that RNA editing is particularly common in behaviorally sophisticated coleoid cephalopods, with tens of thousands of evolutionarily conserved sites. Editing is enriched in the nervous system, affecting molecules pertinent for excitability and neuronal morphology. The genomic sequence flanking editing sites is highly conserved, suggesting that the process confers a selective advantage. Due to the large number of sites, the surrounding conservation greatly reduces the number of mutations and genomic polymorphisms in protein-coding regions. This trade-off between genome evolution and transcriptome plasticity highlights the importance of RNA recoding as a strategy for diversifying proteins, particularly those associated with neural function. PAPERCLIP.
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Evolución Biológica , Cefalópodos/genética , Edición de ARN , Transcriptoma , Adenosina Desaminasa/metabolismo , Secuencia de Aminoácidos , Animales , Cefalópodos/clasificación , Cefalópodos/metabolismo , Sistema Nervioso/metabolismo , Canales de Potasio con Entrada de Voltaje/química , Canales de Potasio con Entrada de Voltaje/genética , Alineación de SecuenciaRESUMEN
Molecular chaperones are critical for protein homeostasis and are implicated in several human pathologies such as neurodegeneration and cancer. While the binding of chaperones to nascent and misfolded proteins has been studied in great detail, the direct interaction between chaperones and RNA has not been systematically investigated. Here, we provide the evidence for widespread interaction between chaperones and RNA in human cells. We show that the major chaperone heat shock protein 70 (HSP70) binds to non-coding RNA transcribed by RNA polymerase III (RNA Pol III) such as tRNA and 5S rRNA. Global chromatin profiling revealed that HSP70 binds genomic sites of transcription by RNA Pol III. Detailed biochemical analyses showed that HSP70 alleviates the inhibitory effect of cognate tRNA transcript on tRNA gene transcription. Thus, our study uncovers an unexpected role of HSP70-RNA interaction in the biogenesis of a specific class of non-coding RNA with wider implications in cancer therapeutics.
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Proteínas HSP70 de Choque Térmico , Neoplasias , Humanos , Proteínas HSP70 de Choque Térmico/genética , Proteínas HSP70 de Choque Térmico/metabolismo , Chaperonas Moleculares/metabolismo , ARN , ARN Polimerasa III/genética , ARN Polimerasa III/metabolismo , ARN de Transferencia/genética , ARN no Traducido/genéticaRESUMEN
Cell diversification is at the base of increasing multicellular organism complexity in phylogeny achieved during ontogeny. However, there are also functions common to all cells, such as cell division, cell migration, translation, endocytosis, exocytosis, etc. Here we revisit the organelles involved in such common functions, reviewing recent evidence of unexpected differences of proteins at these organelles. For instance, centrosomes or mitochondria differ significantly in their protein composition in different, sometimes closely related, cell types. This has relevance for development and disease. Particularly striking is the high amount and diversity of RNA-binding proteins at these and other organelles, which brings us to review the evidence for RNA at different organelles and suborganelles. We include a discussion about (sub)organelles involved in translation, such as the nucleolus and ribosomes, for which unexpected cell type-specific diversity has also been reported. We propose here that the heterogeneity of these organelles and compartments represents a novel mechanism for regulating cell diversity. One reason is that protein functions can be multiplied by their different contributions in distinct organelles, as also exemplified by proteins with moonlighting function. The specialized organelles still perform pan-cellular functions but in a cell type-specific mode, as discussed here for centrosomes, mitochondria, vesicles, and other organelles. These can serve as regulatory hubs for the storage and transport of specific and functionally important regulators. In this way, they can control cell differentiation, plasticity, and survival. We further include examples highlighting the relevance for disease and propose to examine organelles in many more cell types for their possible differences with functional relevance.
Asunto(s)
Mitocondrias , Orgánulos , Orgánulos/metabolismo , Mitocondrias/metabolismo , División Celular , Ribosomas/metabolismo , Diferenciación CelularRESUMEN
Metabolic activity is intimately linked to T cell fate and function. Using high-resolution mass spectrometry, we generated dynamic metabolome and proteome profiles of human primary naive T cells following activation. We discovered critical changes in the arginine metabolism that led to a drop in intracellular L-arginine concentration. Elevating L-arginine levels induced global metabolic changes including a shift from glycolysis to oxidative phosphorylation in activated T cells and promoted the generation of central memory-like cells endowed with higher survival capacity and, in a mouse model, anti-tumor activity. Proteome-wide probing of structural alterations, validated by the analysis of knockout T cell clones, identified three transcriptional regulators (BAZ1B, PSIP1, and TSN) that sensed L-arginine levels and promoted T cell survival. Thus, intracellular L-arginine concentrations directly impact the metabolic fitness and survival capacity of T cells that are crucial for anti-tumor responses.
Asunto(s)
Arginina/metabolismo , Linfocitos T CD4-Positivos/inmunología , Inmunomodulación , Activación de Linfocitos , Melanoma Experimental/inmunología , Neoplasias Cutáneas/inmunología , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Animales , Linfocitos T CD4-Positivos/metabolismo , Proteínas de Unión al ADN/metabolismo , Técnicas de Inactivación de Genes , Glucólisis , Humanos , Memoria Inmunológica , Metaboloma , Ratones , Ratones Endogámicos BALB C , Fosforilación Oxidativa , Proteoma , Factores de Transcripción/metabolismo , Transcripción GenéticaRESUMEN
Transcription of tRNA genes by RNA polymerase III (RNAPIII) is tuned by signaling cascades. The emerging notion of differential tRNA gene regulation implies the existence of additional regulatory mechanisms. However, tRNA gene-specific regulators have not been described. Decoding the local chromatin proteome of a native tRNA gene in yeast revealed reprogramming of the RNAPIII transcription machinery upon nutrient perturbation. Among the dynamic proteins, we identified Fpt1, a protein of unknown function that uniquely occupied RNAPIII-regulated genes. Fpt1 binding at tRNA genes correlated with the efficiency of RNAPIII eviction upon nutrient perturbation and required the transcription factors TFIIIB and TFIIIC but not RNAPIII. In the absence of Fpt1, eviction of RNAPIII was reduced, and the shutdown of ribosome biogenesis genes was impaired upon nutrient perturbation. Our findings provide support for a chromatin-associated mechanism required for RNAPIII eviction from tRNA genes and tuning the physiological response to changing metabolic demands.
Asunto(s)
ARN Polimerasa III , Proteínas de Saccharomyces cerevisiae , ARN Polimerasa III/genética , ARN Polimerasa III/metabolismo , Proteoma/genética , Proteoma/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Cromatina/genética , Cromatina/metabolismo , Regulación Fúngica de la Expresión Génica , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , ARN de Transferencia/genética , ARN de Transferencia/metabolismo , Transcripción GenéticaRESUMEN
The ability of RNAs to form specific contacts with other macromolecules provides an important mechanism for subcellular compartmentalization. Here we describe a suite of hybridization-proximity (HyPro) labeling technologies for unbiased discovery of proteins (HyPro-MS) and transcripts (HyPro-seq) associated with RNAs of interest in genetically unperturbed cells. As a proof of principle, we show that HyPro-MS and HyPro-seq can identify both known and previously unexplored spatial neighbors of the noncoding RNAs 45S, NEAT1, and PNCTR expressed at markedly different levels. Notably, HyPro-seq uncovers an extensive repertoire of incompletely processed, adenosine-to-inosine-edited transcripts accumulating at the interface between their encoding chromosomal regions and the NEAT1-containing paraspeckle compartment. At least some of these targets require NEAT1 for their optimal expression. Overall, this study provides a versatile toolkit for dissecting RNA interactomes in diverse biomedical contexts and expands our understanding of the functional architecture of the mammalian nucleus.
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Compartimento Celular , Núcleo Celular/metabolismo , Técnicas Genéticas , ARN Nuclear/metabolismo , Proteínas de Unión al ARN/metabolismo , Núcleo Celular/genética , Células HeLa , Humanos , Espectrometría de Masas , Prueba de Estudio Conceptual , Unión Proteica , Proteoma , ARN Largo no Codificante/genética , ARN Largo no Codificante/metabolismo , ARN Nuclear/genética , ARN Ribosómico/genética , ARN Ribosómico/metabolismo , Proteínas de Unión al ARN/genética , RNA-Seq , TranscriptomaRESUMEN
The identity of human protein-coding genes is well known, yet our in-depth knowledge of their molecular functions and domain architecture remains limited by shortcomings in homology-based predictions and experimental approaches focused on whole-gene depletion. To bridge this knowledge gap, we developed a method that leverages CRISPR-Cas9-induced mutations across protein-coding genes for the a priori identification of functional regions at the sequence level. As a test case, we applied this method to 48 human mitotic genes, revealing hundreds of regions required for cell proliferation, including domains that were experimentally characterized, ones that were predicted based on homology, and novel ones. We validated screen outcomes for 15 regions, including amino acids 387-402 of Mad1, which were previously uncharacterized but contribute to Mad1 kinetochore localization and chromosome segregation fidelity. Altogether, we demonstrate that CRISPR-Cas9-based tiling mutagenesis identifies key functional domains in protein-coding genes de novo, which elucidates separation of function mutants and allows functional annotation across the human proteome.
Asunto(s)
Sistemas CRISPR-Cas , Sistemas CRISPR-Cas/genética , Humanos , MutagénesisRESUMEN
Mast cells are rare tissue-resident cells of importance to human allergies. To understand the structural basis of principle mast cell functions, we analyzed the proteome of primary human and mouse mast cells by quantitative mass spectrometry. We identified a mast-cell-specific proteome signature, indicative of a unique lineage, only distantly related to other immune cell types, including innate immune cells. Proteome comparison between human and mouse suggested evolutionary conservation of core mast cell functions. In addition to specific proteases and proteins associated with degranulation and proteoglycan biosynthesis, mast cells expressed proteins potentially involved in interactions with neurons and neurotransmitter metabolism, including cell adhesion molecules, ion channels, and G protein coupled receptors. Toward targeted cell ablation in severe allergic diseases, we used MRGPRX2 for mast cell depletion in human skin biopsies. These proteome analyses suggest a unique role of mast cells in the immune system, probably intertwined with the nervous system.
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Mastocitos/citología , Mastocitos/inmunología , Animales , Biomarcadores/metabolismo , Degranulación de la Célula , Linaje de la Célula , Células Cultivadas , Tejido Conectivo/inmunología , Humanos , Inmunoterapia , Mastocitos/metabolismo , Proteínas de la Membrana/inmunología , Proteínas de la Membrana/metabolismo , Ratones , Ratones Endogámicos C57BL , Proteínas del Tejido Nervioso/inmunología , Proteínas del Tejido Nervioso/metabolismo , Neuroinmunomodulación , Proteoglicanos/biosíntesis , Proteoma , Receptores Acoplados a Proteínas G/inmunología , Receptores Acoplados a Proteínas G/metabolismo , Receptores de Neuropéptido/inmunología , Receptores de Neuropéptido/metabolismo , Piel/inmunologíaRESUMEN
mRNA translation is coupled to multiprotein complex assembly in the cytoplasm or to protein delivery into intracellular compartments. Here, by combining systematic RNA immunoprecipitation and single-molecule RNA imaging in yeast, we have provided a complete depiction of the co-translational events involved in the biogenesis of a large multiprotein assembly, the nuclear pore complex (NPC). We report that binary interactions between NPC subunits can be established during translation, in the cytoplasm. Strikingly, the nucleoporins Nup1/Nup2, together with a number of nuclear proteins, are instead translated at nuclear pores, through a mechanism involving interactions between their nascent N-termini and nuclear transport receptors. Uncoupling this co-translational recruitment further triggers the formation of cytoplasmic foci of unassembled polypeptides. Altogether, our data reveal that distinct, spatially segregated modes of co-translational interactions foster the ordered assembly of NPC subunits and that localized translation can ensure the proper delivery of proteins to the pore and the nucleus.
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Proteínas de Complejo Poro Nuclear/genética , Biosíntesis de Proteínas , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Transporte Activo de Núcleo Celular , Citoplasma/genética , Citoplasma/metabolismo , Regulación Fúngica de la Expresión Génica , Carioferinas/genética , Carioferinas/metabolismo , Poro Nuclear/genética , Poro Nuclear/metabolismo , Proteínas de Complejo Poro Nuclear/clasificación , Proteínas de Complejo Poro Nuclear/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/clasificación , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMEN
Substantial efforts are underway to deepen our understanding of human brain morphology, structure, and function using high-resolution imaging as well as high-content molecular profiling technologies. The current work adds to these approaches by providing a comprehensive and quantitative protein expression map of 13 anatomically distinct brain regions covering more than 11,000 proteins. This was enabled by the optimization, characterization, and implementation of a high-sensitivity and high-throughput microflow liquid chromatography timsTOF tandem mass spectrometry system (LC-MS/MS) capable of analyzing more than 2,000 consecutive samples prepared from formalin-fixed paraffin embedded (FFPE) material. Analysis of this proteomic resource highlighted brain region-enriched protein expression patterns and functional protein classes, protein localization differences between brain regions and individual markers for specific areas. To facilitate access to and ease further mining of the data by the scientific community, all data can be explored online in a purpose-built R Shiny app (https://brain-region-atlas.proteomics.ls.tum.de).
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Proteómica , Espectrometría de Masas en Tándem , Humanos , Cromatografía Liquida/métodos , Proteómica/métodos , Adhesión en Parafina/métodos , Espectrometría de Masas en Tándem/métodos , Proteínas/metabolismo , Encéfalo/metabolismo , Proteoma/metabolismoRESUMEN
Regulation of transcription and translation are mechanisms through which genetic variants affect complex traits. Expression quantitative trait locus (eQTL) studies have been more successful at identifying cis-eQTL (within 1 Mb of the transcription start site) than trans-eQTL. Here, we tested the cis component of gene expression for association with observed plasma protein levels to identify cis- and trans-acting genes that regulate protein levels. We used transcriptome prediction models from 49 Genotype-Tissue Expression (GTEx) Project tissues to predict the cis component of gene expression and tested the predicted expression of every gene in every tissue for association with the observed abundance of 3,622 plasma proteins measured in 3,301 individuals from the INTERVAL study. We tested significant results for replication in 971 individuals from the Trans-omics for Precision Medicine (TOPMed) Multi-Ethnic Study of Atherosclerosis (MESA). We found 1,168 and 1,210 cis- and trans-acting associations that replicated in TOPMed (FDR < 0.05) with a median expected true positive rate (π1) across tissues of 0.806 and 0.390, respectively. The target proteins of trans-acting genes were enriched for transcription factor binding sites and autoimmune diseases in the GWAS catalog. Furthermore, we found a higher correlation between predicted expression and protein levels of the same underlying gene (R = 0.17) than observed expression (R = 0.10, p = 7.50 × 10-11). This indicates the cis-acting genetically regulated (heritable) component of gene expression is more consistent across tissues than total observed expression (genetics + environment) and is useful in uncovering the function of SNPs associated with complex traits.
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Proteoma , Transcriptoma , Humanos , Transcriptoma/genética , Proteoma/genética , Herencia Multifactorial , Sitios de Carácter Cuantitativo/genética , Estudio de Asociación del Genoma Completo , Polimorfismo de Nucleótido Simple/genéticaRESUMEN
Transcriptome-wide association study (TWAS) tools have been applied to conduct proteome-wide association studies (PWASs) by integrating proteomics data with genome-wide association study (GWAS) summary data. The genetic effects of PWAS-identified significant genes are potentially mediated through genetically regulated protein abundance, thus informing the underlying disease mechanisms better than GWAS loci. However, existing TWAS/PWAS tools are limited by considering only one statistical model. We propose an omnibus PWAS pipeline to account for multiple statistical models and demonstrate improved performance by simulation and application studies of Alzheimer disease (AD) dementia. We employ the Aggregated Cauchy Association Test to derive omnibus PWAS (PWAS-O) p values from PWAS p values obtained by three existing tools assuming complementary statistical models-TIGAR, PrediXcan, and FUSION. Our simulation studies demonstrated improved power, with well-calibrated type I error, for PWAS-O over all three individual tools. We applied PWAS-O to studying AD dementia with reference proteomic data profiled from dorsolateral prefrontal cortex of postmortem brains from individuals of European ancestry. We identified 43 risk genes, including 5 not identified by previous studies, which are interconnected through a protein-protein interaction network that includes the well-known AD risk genes TOMM40, APOC1, and APOC2. We also validated causal genetic effects mediated through the proteome for 27 (63%) PWAS-O risk genes, providing insights into the underlying biological mechanisms of AD dementia and highlighting promising targets for therapeutic development. PWAS-O can be easily applied to studying other complex diseases.
RESUMEN
Subcellular localization is a main determinant of protein function; however, a global view of cellular proteome organization remains relatively unexplored. We have developed a robust mass spectrometry-based analysis pipeline to generate a proteome-wide view of subcellular localization for proteins mapping to 12,418 individual genes across five cell lines. Based on more than 83,000 unique classifications and correlation profiling, we investigate the effect of alternative splicing and protein domains on localization, complex member co-localization, cell-type-specific localization, as well as protein relocalization after growth factor inhibition. Our analysis provides information about the cellular architecture and complexity of the spatial organization of the proteome; we show that the majority of proteins have a single main subcellular location, that alternative splicing rarely affects subcellular location, and that cell types are best distinguished by expression of proteins exposed to the surrounding environment. The resource is freely accessible via www.subcellbarcode.org.
Asunto(s)
Cromatografía Liquida , Espectrometría de Masas , Proteínas/metabolismo , Proteoma , Proteómica/métodos , Fracciones Subcelulares/metabolismo , Biomarcadores/metabolismo , Fraccionamiento Celular , Biología Computacional , Receptores ErbB/antagonistas & inhibidores , Receptores ErbB/metabolismo , Gefitinib/farmacología , Humanos , Focalización Isoeléctrica , Células MCF-7 , Inhibidores de Proteínas Quinasas/farmacología , Transporte de Proteínas , Proteínas/antagonistas & inhibidores , Proteínas/clasificación , Proteínas/genética , Reproducibilidad de los Resultados , Fracciones Subcelulares/clasificación , Fracciones Subcelulares/efectos de los fármacosRESUMEN
The comprehensive but specific identification of RNA-binding proteins as well as the discovery of RNA-associated protein functions remain major challenges in RNA biology. Here we adapt the concept of RNA dependence, defining a protein as RNA dependent when its interactome depends on RNA. We converted this concept into a proteome-wide, unbiased, and enrichment-free screen called R-DeeP (RNA-dependent proteins), based on density gradient ultracentrifugation. Quantitative mass spectrometry identified 1,784 RNA-dependent proteins, including 537 lacking known links to RNA. Exploiting the quantitative nature of R-DeeP, proteins were classified as not, partially, or completely RNA dependent. R-DeeP identified the transcription factor CTCF as completely RNA dependent, and we uncovered that RNA is required for the CTCF-chromatin association. Additionally, R-DeeP allows reconstruction of protein complexes based on co-segregation. The whole dataset is available at http://R-DeeP.dkfz.de, providing proteome-wide, specific, and quantitative identification of proteins with RNA-dependent interactions and aiming at future functional discovery of RNA-protein complexes.