RESUMEN
The systematic determination of protein function is a key goal of modern biology, but remains challenging with current approaches. Here we present ORFtag, a versatile, cost-effective and highly efficient method for the massively parallel tagging and functional interrogation of proteins at the proteome scale. ORFtag uses retroviral vectors bearing a promoter, peptide tag and splice donor to generate fusions between the tag and endogenous open reading frames (ORFs). We demonstrate the utility of ORFtag through functional screens for transcriptional activators, repressors and posttranscriptional regulators in mouse embryonic stem cells. Each screen recovers known and identifies new regulators, including long ORFs inaccessible by other methods. Among other hits, we find that Zfp574 is a highly selective transcriptional activator and that oncogenic fusions often function as transactivators.
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Sistemas de Lectura Abierta , Proteoma , Animales , Ratones , Proteoma/metabolismo , Células Madre Embrionarias de Ratones/metabolismo , Proteómica/métodos , HumanosRESUMEN
The maternal-to-zygotic transition (MZT) is a key developmental process in metazoan embryos that involves the activation of zygotic transcription (ZGA) and degradation of maternal transcripts. We employed metabolic mRNA sequencing (SLAMseq) to deconvolute the compound embryonic transcriptome in zebrafish. While mitochondrial zygotic transcripts prevail prior to MZT, we uncover the spurious transcription of hundreds of short and intron-poor genes as early as the 2-cell stage. Upon ZGA, most zygotic transcripts originate from thousands of maternal-zygotic (MZ) genes that are transcribed at rates comparable to those of hundreds of purely zygotic genes and replenish maternal mRNAs at distinct timescales. Rapid replacement of MZ transcripts involves transcript decay features unrelated to major maternal degradation pathways and promotes de novo synthesis of the core gene expression machinery by increasing poly(A)-tail length and translation efficiency. SLAMseq hence provides insights into the timescales, molecular features, and regulation of MZT during zebrafish embryogenesis.
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Desarrollo Embrionario , Pez Cebra , Animales , Pez Cebra/metabolismo , Desarrollo Embrionario/genética , Cigoto/metabolismo , ARN Mensajero/metabolismo , Transcriptoma/genética , Regulación del Desarrollo de la Expresión GénicaRESUMEN
Most endogenous retroviruses (ERVs) in mammals are incapable of retrotransposition; therefore, why ERV derepression is associated with lethality during early development has been a mystery. Here, we report that rapid and selective degradation of the heterochromatin adapter protein TRIM28 triggers dissociation of transcriptional condensates from loci encoding super-enhancer (SE)-driven pluripotency genes and their association with transcribed ERV loci in murine embryonic stem cells. Knockdown of ERV RNAs or forced expression of SE-enriched transcription factors rescued condensate localization at SEs in TRIM28-degraded cells. In a biochemical reconstitution system, ERV RNA facilitated partitioning of RNA polymerase II and the Mediator coactivator into phase-separated droplets. In TRIM28 knockout mouse embryos, single-cell RNA-seq analysis revealed specific depletion of pluripotent lineages. We propose that coding and noncoding nascent RNAs, including those produced by retrotransposons, may facilitate 'hijacking' of transcriptional condensates in various developmental and disease contexts.
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Retrovirus Endógenos , Animales , Células Madre Embrionarias , Retrovirus Endógenos/genética , Heterocromatina , Mamíferos/genética , Ratones , Cuerpos Nucleares , RetroelementosRESUMEN
Gene expression is controlled at multiple levels, including RNA transcription and turnover. But determining the relative contributions of RNA biogenesis and decay to the steady-state abundance of cellular transcripts remains challenging because conventional transcriptomics approaches do not provide the temporal resolution to derive the kinetic parameters underlying steady-state gene expression.Here, we describe a protocol that combines metabolic RNA labeling by 4-thiouridine with chemical nucleoside conversion and whole-transcriptome sequencing followed by bioinformatics analysis to determine RNA stability in cultured cells at a genomic scale. Time-resolved transcriptomics by thiol (SH)-linked alkylation for the metabolic sequencing of RNA (SLAMseq) provides accurate information on transcript half-lives across annotated features in the genome, including by-products of transcription, such as introns. We provide a step-by-step instruction for time-resolved transcriptomics, which enhances traditional RNA sequencing protocols to acquire the temporal resolution required to directly measure the cellular kinetics of RNA turnover under physiological conditions.
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Estabilidad del ARN , Transcriptoma , Perfilación de la Expresión Génica , ARN/genética , Análisis de Secuencia de ARN , TiouridinaRESUMEN
Nuclear Argonaute proteins, guided by their bound small RNAs to nascent target transcripts, mediate cotranscriptional silencing of transposons and repetitive genomic loci through heterochromatin formation. The molecular mechanisms involved in this process are incompletely understood. Here, we show that the SFiNX complex, a silencing mediator downstream from nuclear Piwi-piRNA complexes in Drosophila, facilitates cotranscriptional silencing as a homodimer. The dynein light chain protein Cut up/LC8 mediates SFiNX dimerization, and its function can be bypassed by a heterologous dimerization domain, arguing for a constitutive SFiNX dimer. Dimeric, but not monomeric SFiNX, is capable of forming molecular condensates in a nucleic acid-stimulated manner. Mutations that prevent SFiNX dimerization result in loss of condensate formation in vitro and the inability of Piwi to initiate heterochromatin formation and silence transposons in vivo. We propose that multivalent SFiNX-nucleic acid interactions are critical for heterochromatin establishment at piRNA target loci in a cotranscriptional manner.
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Proteínas Argonautas/metabolismo , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/genética , Regulación del Desarrollo de la Expresión Génica/genética , Silenciador del Gen/fisiología , Complejos Multiproteicos/metabolismo , Animales , Dimerización , Proteínas de Drosophila/química , Drosophila melanogaster/metabolismo , Dineínas/metabolismo , Complejos Multiproteicos/química , Complejos Multiproteicos/genética , Proteínas Nucleares/química , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Proteínas de Transporte Nucleocitoplasmático/química , Proteínas de Transporte Nucleocitoplasmático/genética , Proteínas de Transporte Nucleocitoplasmático/metabolismo , Subunidades de Proteína/genética , Subunidades de Proteína/metabolismo , Proteínas de Unión al ARN/química , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismoRESUMEN
The varying rates at which mRNAs decay are tightly coordinated with transcriptional changes to shape gene expression during development and disease. But currently available RNA sequencing approaches lack the temporal information to determine the relative contribution of RNA biogenesis, processing and turnover to the establishment of steady-state gene expression profiles.Here, we describe a protocol that combines metabolic RNA labeling with chemical nucleoside conversion by thiol-linked alkylation of 4-thiouridine to determine RNA stability in cultured cells (SLAMseq). When coupled to cost-effective mRNA 3' end sequencing approaches, SLAMseq determines the half-life of polyadenylated transcripts in a global and transcript-specific manner using untargeted or targeted cDNA library preparation protocols.We provide a step-by-step instruction for time-resolved mRNA 3' end sequencing, which augments traditional RNA-seq approaches to acquire the temporal resolution necessary to study the molecular principles that control gene expression.
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Nucleósidos/genética , Estabilidad del ARN/genética , ARN Mensajero/genética , Alquilación/genética , Animales , Línea Celular , Biblioteca de Genes , Ratones , Tiouridina/metabolismo , Transcriptoma/genéticaRESUMEN
Argonaute-bound microRNAs silence mRNA expression in a dynamic and regulated manner to control organismal development, physiology, and disease. We employed metabolic small RNA sequencing for a comprehensive view on intracellular microRNA kinetics in Drosophila. Based on absolute rate of biogenesis and decay, microRNAs rank among the fastest produced and longest-lived cellular transcripts, disposing up to 105 copies per cell at steady-state. Mature microRNAs are produced within minutes, revealing tight intracellular coupling of biogenesis that is selectively disrupted by pre-miRNA-uridylation. Control over Argonaute protein homeostasis generates a kinetic bottleneck that cooperates with non-coding RNA surveillance to ensure faithful microRNA loading. Finally, regulated small RNA decay enables the selective rapid turnover of Ago1-bound microRNAs, but not of Ago2-bound small interfering RNAs (siRNAs), reflecting key differences in the robustness of small RNA silencing pathways. Time-resolved small RNA sequencing opens new experimental avenues to deconvolute the timescales, molecular features, and regulation of small RNA silencing pathways in living cells.
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Proteínas Argonautas/metabolismo , Proteínas de Drosophila/metabolismo , Homeostasis/fisiología , MicroARNs/metabolismo , Análisis de Secuencia de ARN , Animales , Proteínas Argonautas/genética , Línea Celular , Proteínas de Drosophila/genética , Drosophila melanogaster , MicroARNs/genéticaRESUMEN
BACKGROUND: Melanocortin 1 Receptor (MC1R) is expressed in a majority of melanoma biopsies and cell lines. We previously demonstrated that three hydrophobic low-affinity HLA-A2-restricted MC1R-derived peptides: MC1R291-298, MC1R244-252 and MC1R283-291 can elicit cytotoxic T-lymphocytes (CTL) responses from normal donor peripheral blood lymphocytes (PBL). Moreover, peptide-specific CTL recognized a panel of MHC-matched melanomas, demonstrating that human melanoma cell lines naturally present MC1R epitopes. However, the natural presence of MC1R-specific T cells in melanoma patient's tumour and blood remains unknown. METHODS: The presence of anti-MC1R specific CD8(+) T cells was established in a population of melanoma-specific T cells derived from peripheral blood mononuclear cells (PBMC) and tumour-infiltrating lymphocytes (TIL) from HLA-A2(+) melanoma patients. RESULTS: CTLs specific for the three MC1R-derived peptides that lysed allogeneic HLA-A2(+)MC1R(+) melanomas were elicited from PBMC, demonstrating the existence of an anti-MC1R T cell repertoire in melanoma patients. Moreover, TILs also recognized MC1R epitopes and HLA-A2(+) melanoma cell lines. Finally, HLA-A2/MC1R244-specific CD8(+) T cell clones derived from TILs and a subset of MC1R291 specific TILs were identified using HLA-A2/MC1R tetramers. CONCLUSION: Our results demonstrate that MC1R-derived peptides are common immunogenic epitopes for melanoma-specific CTLs and TILs, and may thus be useful for the development of anti-melanoma immunotherapy.