RESUMEN
Vault RNAs (vtRNA) are small non-coding RNAs transcribed by RNA polymerase III found in many eukaryotes. Although they have been linked to drug resistance, apoptosis, and viral replication, their molecular functions remain unclear. Here, we show that vault RNAs directly bind the autophagy receptor sequestosome-1/p62 in human and murine cells. Overexpression of human vtRNA1-1 inhibits, while its antisense LNA-mediated knockdown enhances p62-dependent autophagy. Starvation of cells reduces the steady-state and p62-bound levels of vault RNA1-1 and induces autophagy. Mechanistically, p62 mutants that fail to bind vtRNAs display increased p62 homo-oligomerization and augmented interaction with autophagic effectors. Thus, vtRNA1-1 directly regulates selective autophagy by binding p62 and interference with oligomerization, a critical step of p62 function. Our data uncover a striking example of the potential of RNA to control protein functions directly, as previously recognized for protein-protein interactions and post-translational modifications.
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Autofagia/genética , Partículas Ribonucleoproteicas en Bóveda/genética , Partículas Ribonucleoproteicas en Bóveda/metabolismo , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Animales , Línea Celular , Células HeLa , Humanos , Ratones , Células RAW 264.7 , ARN/metabolismo , ARN no Traducido/metabolismo , ARN no Traducido/fisiología , Proteína Sequestosoma-1/genética , Proteína Sequestosoma-1/metabolismoRESUMEN
Mouse embryonic stem cells (ESCs) are maintained in a naive ground state of pluripotency in the presence of MEK and GSK3 inhibitors. Here, we show that ground-state ESCs express low Myc levels. Deletion of both c-myc and N-myc (dKO) or pharmacological inhibition of Myc activity strongly decreases transcription, splicing, and protein synthesis, leading to proliferation arrest. This process is reversible and occurs without affecting pluripotency, suggesting that Myc-depleted stem cells enter a state of dormancy similar to embryonic diapause. Indeed, c-Myc is depleted in diapaused blastocysts, and the differential expression signatures of dKO ESCs and diapaused epiblasts are remarkably similar. Following Myc inhibition, pre-implantation blastocysts enter biosynthetic dormancy but can progress through their normal developmental program after transfer into pseudo-pregnant recipients. Our study shows that Myc controls the biosynthetic machinery of stem cells without affecting their potency, thus regulating their entry and exit from the dormant state.
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Células Madre Embrionarias/citología , Genes myc , Proteínas Proto-Oncogénicas c-myc/genética , Animales , Blastocisto/metabolismo , Proliferación Celular , Embrión de Mamíferos/citología , Embrión de Mamíferos/metabolismo , Células Madre Embrionarias/metabolismo , Femenino , Técnicas de Inactivación de Genes , Masculino , Ratones , Ratones Endogámicos C57BLRESUMEN
Deciphering the impact of genetic variants on gene regulation is fundamental to understanding human disease. Although gene regulation often involves long-range interactions, it is unknown to what extent non-coding genetic variants influence distal molecular phenotypes. Here, we integrate chromatin profiling for three histone marks in lymphoblastoid cell lines (LCLs) from 75 sequenced individuals with LCL-specific Hi-C and ChIA-PET-based chromatin contact maps to uncover one of the largest collections of local and distal histone quantitative trait loci (hQTLs). Distal QTLs are enriched within topologically associated domains and exhibit largely concordant variation of chromatin state coordinated by proximal and distal non-coding genetic variants. Histone QTLs are enriched for common variants associated with autoimmune diseases and enable identification of putative target genes of disease-associated variants from genome-wide association studies. These analyses provide insights into how genetic variation can affect human disease phenotypes by coordinated changes in chromatin at interacting regulatory elements.
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Cromatina/metabolismo , Cromosomas Humanos/metabolismo , Proyecto Genoma Humano , Línea Celular , Cromosomas Humanos/química , Estudios de Cohortes , Femenino , Redes Reguladoras de Genes , Estudio de Asociación del Genoma Completo , Histonas/metabolismo , Humanos , Linfocitos/metabolismo , Masculino , Sitios de Carácter Cuantitativo , Elementos Reguladores de la TranscripciónRESUMEN
Selective protein degradation by the ubiquitin-proteasome system (UPS) is involved in all cellular processes. However, the substrates and specificity of most UPS components are not well understood. Here we systematically characterized the UPS in Saccharomyces cerevisiae. Using fluorescent timers, we determined how loss of individual UPS components affects yeast proteome turnover, detecting phenotypes for 76% of E2, E3, and deubiquitinating enzymes. We exploit this dataset to gain insights into N-degron pathways, which target proteins carrying N-terminal degradation signals. We implicate Ubr1, an E3 of the Arg/N-degron pathway, in targeting mitochondrial proteins processed by the mitochondrial inner membrane protease. Moreover, we identify Ylr149c/Gid11 as a substrate receptor of the glucose-induced degradation-deficient (GID) complex, an E3 of the Pro/N-degron pathway. Our results suggest that Gid11 recognizes proteins with N-terminal threonines, expanding the specificity of the GID complex. This resource of potential substrates and relationships between UPS components enables exploring functions of selective protein degradation.
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Proteínas Mitocondriales/genética , Complejo de la Endopetidasa Proteasomal/metabolismo , Procesamiento Proteico-Postraduccional , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Ubiquitina-Proteína Ligasas/genética , Perfilación de la Expresión Génica , Regulación Fúngica de la Expresión Génica , Genes Reporteros , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Proteínas Luminiscentes/genética , Proteínas Luminiscentes/metabolismo , Proteínas Mitocondriales/clasificación , Proteínas Mitocondriales/metabolismo , Transporte de Proteínas , Proteolisis , Proteómica/métodos , Saccharomyces cerevisiae/enzimología , Proteínas de Saccharomyces cerevisiae/metabolismo , Treonina/metabolismo , Ubiquitina/genética , Ubiquitina/metabolismo , Ubiquitina-Proteína Ligasas/clasificación , Ubiquitina-Proteína Ligasas/metabolismo , Ubiquitinación , Proteína Fluorescente RojaRESUMEN
Dysfunction of ENaC, the epithelial sodium channel that regulates salt and water reabsorption in epithelia, causes several human diseases, including cystic fibrosis (CF). To develop a global understanding of molecular regulators of ENaC traffic/function and to identify of candidate CF drug targets, we performed a large-scale screen combining high-content live-cell microscopy and siRNAs in human airway epithelial cells. Screening over 6,000 genes identified over 1,500 candidates, evenly divided between channel inhibitors and activators. Genes in the phosphatidylinositol pathway were enriched on the primary candidate list, and these, along with other ENaC activators, were examined further with secondary siRNA validation. Subsequent detailed investigation revealed ciliary neurotrophic factor receptor (CNTFR) as an ENaC modulator and showed that inhibition of (diacylglycerol kinase, iota) DGKι, a protein involved in PiP2 metabolism, downgrades ENaC activity, leading to normalization of both Na+ and fluid absorption in CF airways to non-CF levels in primary human lung cells from CF patients.
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Fibrosis Quística/tratamiento farmacológico , Terapia Molecular Dirigida , Línea Celular , Células Cultivadas , Canales Epiteliales de Sodio/metabolismo , Humanos , Pulmón/citología , Pulmón/metabolismo , ARN Interferente PequeñoRESUMEN
Spatially resolved omics technologies are transforming our understanding of biological tissues. However, the handling of uni- and multimodal spatial omics datasets remains a challenge owing to large data volumes, heterogeneity of data types and the lack of flexible, spatially aware data structures. Here we introduce SpatialData, a framework that establishes a unified and extensible multiplatform file-format, lazy representation of larger-than-memory data, transformations and alignment to common coordinate systems. SpatialData facilitates spatial annotations and cross-modal aggregation and analysis, the utility of which is illustrated in the context of multiple vignettes, including integrative analysis on a multimodal Xenium and Visium breast cancer study.
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Expression of tissue-restricted self antigens (TRAs) in medullary thymic epithelial cells (mTECs) is essential for the induction of self-tolerance and prevents autoimmunity, with each TRA being expressed in only a few mTECs. How this process is regulated in single mTECs and is coordinated at the population level, such that the varied single-cell patterns add up to faithfully represent TRAs, is poorly understood. Here we used single-cell RNA sequencing and obtained evidence of numerous recurring TRA-co-expression patterns, each present in only a subset of mTECs. Co-expressed genes clustered in the genome and showed enhanced chromatin accessibility. Our findings characterize TRA expression in mTECs as a coordinated process that might involve local remodeling of chromatin and thus ensures a comprehensive representation of the immunological self.
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Autoantígenos/genética , Células Epiteliales/inmunología , Regulación de la Expresión Génica/inmunología , ARN Mensajero/metabolismo , Autotolerancia/inmunología , Timo/inmunología , Animales , Autoinmunidad/inmunología , Ensamble y Desensamble de Cromatina , Células Epiteliales/metabolismo , Perfilación de la Expresión Génica , Humanos , Ratones , Autotolerancia/genética , Análisis de la Célula Individual , Timo/citología , Timo/metabolismoRESUMEN
The count table, a numeric matrix of genes × cells, is the basic input data structure in the analysis of single-cell RNA-sequencing data. A common preprocessing step is to adjust the counts for variable sampling efficiency and to transform them so that the variance is similar across the dynamic range. These steps are intended to make subsequent application of generic statistical methods more palatable. Here, we describe four transformation approaches based on the delta method, model residuals, inferred latent expression state and factor analysis. We compare their strengths and weaknesses and find that the latter three have appealing theoretical properties; however, in benchmarks using simulated and real-world data, it turns out that a rather simple approach, namely, the logarithm with a pseudo-count followed by principal-component analysis, performs as well or better than the more sophisticated alternatives. This result highlights limitations of current theoretical analysis as assessed by bottom-line performance benchmarks.
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Análisis de Expresión Génica de una Sola Célula , Programas Informáticos , Análisis de Secuencia de ARN/métodos , Análisis de la Célula Individual/métodos , Perfilación de la Expresión Génica/métodosRESUMEN
Organisms use organic molecules called osmolytes to adapt to environmental conditions. In vitro studies indicate that osmolytes thermally stabilize proteins, but mechanisms are controversial, and systematic studies within the cellular milieu are lacking. We analyzed Escherichia coli and human protein thermal stabilization by osmolytes in situ and across the proteome. Using structural proteomics, we probed osmolyte effects on protein thermal stability, structure and aggregation, revealing common mechanisms but also osmolyte- and protein-specific effects. All tested osmolytes (trimethylamine N-oxide, betaine, glycerol, proline, trehalose and glucose) stabilized many proteins, predominantly via a preferential exclusion mechanism, and caused an upward shift in temperatures at which most proteins aggregated. Thermal profiling of the human proteome provided evidence for intrinsic disorder in situ but also identified potential structure in predicted disordered regions. Our analysis provides mechanistic insight into osmolyte function within a complex biological matrix and sheds light on the in situ prevalence of intrinsically disordered regions.
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Escherichia coli , Estabilidad Proteica , Proteoma , Proteoma/metabolismo , Proteoma/química , Humanos , Escherichia coli/metabolismo , Temperatura , Betaína/química , Betaína/metabolismo , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Trehalosa/química , Trehalosa/metabolismo , Proteómica/métodos , Prolina/química , Prolina/metabolismo , Glucosa/química , Glucosa/metabolismo , Glicerol/química , Glicerol/metabolismo , MetilaminasRESUMEN
Enhanced crosslinking and immunoprecipitation (eCLIP) sequencing is a method for transcriptome-wide detection of binding sites of RNA-binding proteins (RBPs). However, identified crosslink sites can deviate from experimentally established functional elements of even well-studied RBPs. Current peak-calling strategies result in low replication and high false positive rates. Here, we present the R/Bioconductor package DEWSeq that makes use of replicate information and size-matched input controls. We benchmarked DEWSeq on 107 RBPs for which both eCLIP data and RNA sequence motifs are available and were able to more than double the number of motif-containing binding regions relative to standard eCLIP processing. The improvement not only relates to the number of binding sites (3.1-fold with known motifs for RBFOX2), but also their subcellular localization (1.9-fold of mitochondrial genes for FASTKD2) and structural targets (2.2-fold increase of stem-loop regions for SLBP. On several orthogonal CLIP-seq datasets, DEWSeq recovers a larger number of motif-containing binding sites (3.3-fold). DEWSeq is a well-documented R/Bioconductor package, scalable to adequate numbers of replicates, and tends to substantially increase the proportion and total number of RBP binding sites containing biologically relevant features.
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Proteínas de Unión al ARN , Programas Informáticos , Sitios de Unión , Inmunoprecipitación , Unión Proteica , ARN/química , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismoRESUMEN
The complexity of the functional proteome extends considerably beyond the coding genome, resulting in millions of proteoforms. Investigation of proteoforms and their functional roles is important to understand cellular physiology and its deregulation in diseases but challenging to perform systematically. Here we applied thermal proteome profiling with deep peptide coverage to detect functional proteoform groups in acute lymphoblastic leukemia cell lines with different cytogenetic aberrations. We detected 15,846 proteoforms, capturing differently spliced, cleaved and post-translationally modified proteins expressed from 9,290 genes. We identified differential co-aggregation of proteoform pairs and established links to disease biology. Moreover, we systematically made use of measured biophysical proteoform states to find specific biomarkers of drug sensitivity. Our approach, thus, provides a powerful and unique tool for systematic detection and functional annotation of proteoform groups.
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Proteoma , Espectrometría de Masas en Tándem , Proteoma/metabolismo , Espectrometría de Masas en Tándem/métodos , Línea CelularRESUMEN
The evolution of gene expression in mammalian organ development remains largely uncharacterized. Here we report the transcriptomes of seven organs (cerebrum, cerebellum, heart, kidney, liver, ovary and testis) across developmental time points from early organogenesis to adulthood for human, rhesus macaque, mouse, rat, rabbit, opossum and chicken. Comparisons of gene expression patterns identified correspondences of developmental stages across species, and differences in the timing of key events during the development of the gonads. We found that the breadth of gene expression and the extent of purifying selection gradually decrease during development, whereas the amount of positive selection and expression of new genes increase. We identified differences in the temporal trajectories of expression of individual genes across species, with brain tissues showing the smallest percentage of trajectory changes, and the liver and testis showing the largest. Our work provides a resource of developmental transcriptomes of seven organs across seven species, and comparative analyses that characterize the development and evolution of mammalian organs.
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Regulación del Desarrollo de la Expresión Génica , Organogénesis/genética , Transcriptoma/genética , Animales , Evolución Biológica , Pollos/genética , Femenino , Humanos , Macaca mulatta/genética , Masculino , Ratones , Zarigüeyas/genética , Conejos , RatasRESUMEN
MOTIVATION: Multiple factors can impact accuracy and reproducibility of mass spectrometry data. There is a need to integrate quality assessment and control into data analytic workflows. RESULTS: The MsQuality package calculates 43 low-level quality metrics based on the controlled mzQC vocabulary defined by the HUPO-PSI on a single mass spectrometry-based measurement of a sample. It helps to identify low-quality measurements and track data quality. Its use of community-standard quality metrics facilitates comparability of quality assessment and control (QA/QC) criteria across datasets. AVAILABILITY AND IMPLEMENTATION: The R package MsQuality is available through Bioconductor at https://bioconductor.org/packages/MsQuality.
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Programas Informáticos , Reproducibilidad de los Resultados , Espectrometría de Masas/métodosRESUMEN
SUMMARY: Transcriptome-wide detection of binding sites of RNA-binding proteins is achieved using Individual-nucleotide crosslinking and immunoprecipitation (iCLIP) and its derivative enhanced CLIP (eCLIP) sequencing methods. Here, we introduce htseq-clip, a python package developed for preprocessing, extracting and summarizing crosslink site counts from i/eCLIP experimental data. The package delivers crosslink site count matrices along with other metrics, which can be directly used for filtering and downstream analyses such as the identification of differential binding sites. AVAILABILITY AND IMPLEMENTATION: The Python package htseq-clip is available via pypi (python package index), bioconda and the Galaxy Tool Shed under the open source MIT License. The code is hosted at https://github.com/EMBL-Hentze-group/htseq-clip and documentation is available under https://htseq-clip.readthedocs.io/en/latest.
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Programas Informáticos , Transcriptoma , Sitios de Unión , Proteínas de Unión al ARN/metabolismo , InmunoprecipitaciónRESUMEN
Understanding the interplay of the proteome and the metabolome helps to understand cellular regulation and response. To enable robust inferences from such multi-omics analyses, we introduced and evaluated a workflow for combined proteome and metabolome analysis starting from a single sample. Specifically, we integrated established and individually optimized protocols for metabolomic and proteomic profiling (EtOH/MTBE and autoSP3, respectively) into a unified workflow (termed MTBE-SP3), and took advantage of the fact that the protein residue of the metabolomic sample can be used as a direct input for proteome analysis. We particularly evaluated the performance of proteome analysis in MTBE-SP3, and demonstrated equivalence of proteome profiles irrespective of prior metabolite extraction. In addition, MTBE-SP3 combines the advantages of EtOH/MTBE and autoSP3 for semi-automated metabolite extraction and fully automated proteome sample preparation, respectively, thus advancing standardization and scalability for large-scale studies. We showed that MTBE-SP3 can be applied to various biological matrices (FFPE tissue, fresh-frozen tissue, plasma, serum and cells) to enable implementation in a variety of clinical settings. To demonstrate applicability, we applied MTBE-SP3 and autoSP3 to a lung adenocarcinoma cohort showing consistent proteomic alterations between tumour and non-tumour adjacent tissue independent of the method used. Integration with metabolomic data obtained from the same samples revealed mitochondrial dysfunction in tumour tissue through deregulation of OGDH, SDH family enzymes and PKM. In summary, MTBE-SP3 enables the facile and reliable parallel measurement of proteins and metabolites obtained from the same sample, benefiting from reduced sample variation and input amount. This workflow is particularly applicable for studies with limited sample availability and offers the potential to enhance the integration of metabolomic and proteomic datasets.
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Reversible protein phosphorylation is an important mechanism for regulating (dis)assembly of biomolecular condensates. However, condensate-specific phosphosites remain largely unknown, thereby limiting our understanding of the underlying mechanisms. Here, we combine solubility proteome profiling with phosphoproteomics to quantitatively map several hundred phosphosites enriched in either soluble or condensate-bound protein subpopulations, including a subset of phosphosites modulating protein-RNA interactions. We show that multi-phosphorylation of the C-terminal disordered segment of heteronuclear ribonucleoprotein A1 (HNRNPA1), a key RNA-splicing factor, reduces its ability to locate to nuclear clusters. For nucleophosmin 1 (NPM1), an essential nucleolar protein, we show that phosphorylation of S254 and S260 is crucial for lowering its partitioning to the nucleolus and additional phosphorylation of distal sites enhances its retention in the nucleoplasm. These phosphorylation events decrease RNA and protein interactions of NPM1 to regulate its condensation. Our dataset is a rich resource for systematically uncovering the phosphoregulation of biomolecular condensates.
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Condensados Biomoleculares , Proteoma , Proteínas Nucleares/metabolismo , Fosforilación , Proteoma/metabolismo , ARN/metabolismo , Factores de Empalme de ARN/metabolismo , Ribonucleoproteínas/metabolismoRESUMEN
DNA replication is temporally and spatially organized in all eukaryotes, yet the molecular control and biological function of the replication-timing program are unclear. Rif1 is required for normal genome-wide regulation of replication timing, but its molecular function is poorly understood. Here we show that in mouse embryonic stem cells, Rif1 coats late-replicating domains and, with Lamin B1, identifies most of the late-replicating genome. Rif1 is an essential determinant of replication timing of non-Lamin B1-bound late domains. We further demonstrate that Rif1 defines and restricts the interactions between replication-timing domains during the G1 phase, thereby revealing a function of Rif1 as organizer of nuclear architecture. Rif1 loss affects both number and replication-timing specificity of the interactions between replication-timing domains. In addition, during the S phase, Rif1 ensures that replication of interacting domains is temporally coordinated. In summary, our study identifies Rif1 as the molecular link between nuclear architecture and replication-timing establishment in mammals.
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Núcleo Celular/metabolismo , Momento de Replicación del ADN , Proteínas de Unión a Telómeros/metabolismo , Animales , Proliferación Celular , Cromatina/metabolismo , Inmunoprecipitación de Cromatina , Islas de CpG/genética , Fase G1 , Eliminación de Gen , Regulación de la Expresión Génica , Ratones , Células Madre Embrionarias de Ratones/citología , Células Madre Embrionarias de Ratones/metabolismo , Unión Proteica , Estructura Terciaria de Proteína , Proteínas de Unión a Telómeros/química , Sitio de Iniciación de la TranscripciónRESUMEN
Recent technological advancements have enabled the profiling of a large number of genome-wide features in individual cells. However, single-cell data present unique challenges that require the development of specialized methods and software infrastructure to successfully derive biological insights. The Bioconductor project has rapidly grown to meet these demands, hosting community-developed open-source software distributed as R packages. Featuring state-of-the-art computational methods, standardized data infrastructure and interactive data visualization tools, we present an overview and online book (https://osca.bioconductor.org) of single-cell methods for prospective users.
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Análisis de la Célula Individual/métodos , Perfilación de la Expresión Génica , Genoma , Secuenciación de Nucleótidos de Alto Rendimiento , Programas InformáticosRESUMEN
An amendment to this paper has been published and can be accessed via a link at the top of the paper.
RESUMEN
MOTIVATION: First-line data quality assessment and exploratory data analysis are integral parts of any data analysis workflow. In high-throughput quantitative omics experiments (e.g. transcriptomics, proteomics and metabolomics), after initial processing, the data are typically presented as a matrix of numbers (feature IDs × samples). Efficient and standardized data quality metrics calculation and visualization are key to track the within-experiment quality of these rectangular data types and to guarantee for high-quality datasets and subsequent biological question-driven inference. RESULTS: We present MatrixQCvis, which provides interactive visualization of data quality metrics at the per-sample and per-feature level using R's shiny framework. It provides efficient and standardized ways to analyze data quality of quantitative omics data types that come in a matrix-like format (features IDs × samples). MatrixQCvis builds upon the Bioconductor SummarizedExperiment S4 class and thus facilitates the integration into existing workflows. AVAILABILITY AND IMPLEMENTATION: MatrixQCVis is implemented in R. It is available via Bioconductor and released under the GPL v3.0 license. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.