RESUMO
Many proteins regulate the expression of genes by binding to specific regions encoded in the genome1. Here we introduce a new data set of RNA elements in the human genome that are recognized by RNA-binding proteins (RBPs), generated as part of the Encyclopedia of DNA Elements (ENCODE) project phase III. This class of regulatory elements functions only when transcribed into RNA, as they serve as the binding sites for RBPs that control post-transcriptional processes such as splicing, cleavage and polyadenylation, and the editing, localization, stability and translation of mRNAs. We describe the mapping and characterization of RNA elements recognized by a large collection of human RBPs in K562 and HepG2 cells. Integrative analyses using five assays identify RBP binding sites on RNA and chromatin in vivo, the in vitro binding preferences of RBPs, the function of RBP binding sites and the subcellular localization of RBPs, producing 1,223 replicated data sets for 356 RBPs. We describe the spectrum of RBP binding throughout the transcriptome and the connections between these interactions and various aspects of RNA biology, including RNA stability, splicing regulation and RNA localization. These data expand the catalogue of functional elements encoded in the human genome by the addition of a large set of elements that function at the RNA level by interacting with RBPs.
Assuntos
Proteínas de Ligação a RNA/química , Proteínas de Ligação a RNA/metabolismo , Transcriptoma/genética , Processamento Alternativo/genética , Sequência de Bases , Sítios de Ligação , Linhagem Celular , Cromatina/genética , Cromatina/metabolismo , Bases de Dados Genéticas , Feminino , Técnicas de Silenciamento de Genes , Humanos , Espaço Intracelular/genética , Masculino , Ligação Proteica , RNA Mensageiro/química , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/genética , Especificidade por SubstratoRESUMO
The subcellular trafficking of RNA molecules is a conserved feature of eukaryotic cells and plays key functions in diverse processes implicating polarised cellular activities. Large-scale imaging and subcellular transcriptomic studies suggest that regulated RNA localization is a highly prevalent process that appears to be disrupted in several neuromuscular disorders. These features underline the importance and usefulness of implementing procedures to assess global transcriptome subcellular distribution properties. Here, we present a method combining biochemical fractionation of cells and high-throughput RNA sequencing (CeFra-seq) that enables rapid and efficient systematic mapping of RNA cytotopic distributions in cells. The described procedure involves biochemical fractionation to derive extracts of nuclear, cytosolic, endomembrane, cytoplasmic insoluble and extracellular material from cell culture lines. The RNA content of each fraction can then be profiled by deep-sequencing, revealing global subcellular signatures. We provide a detailed protocol for the CeFra-seq procedure along with relevant validation steps and data analysis guidelines to graphically represent RNA spatial distribution features. As a complement to imaging approaches, CeFra-seq represents a powerful and scalable tool to investigate global alterations in RNA trafficking.
Assuntos
Núcleo Celular/genética , Mapeamento Cromossômico/métodos , Citoplasma/genética , Sequenciamento de Nucleotídeos em Larga Escala/métodos , RNA/genética , Análise de Sequência de RNA/métodos , Animais , Fracionamento Celular , Núcleo Celular/metabolismo , Citoplasma/metabolismo , Drosophila , Humanos , Células K562 , RNA/isolamento & purificaçãoRESUMO
In Drosophila melanogaster there are two genes encoding ribosomal protein S5, RpS5a and RpS5b. Here, we demonstrate that RpS5b is required for oogenesis. Females lacking RpS5b produce ovaries with numerous developmental defects that undergo widespread apoptosis in mid-oogenesis. Females lacking germline RpS5a are fully fertile, but germline expression of interfering RNA targeting germline RpS5a in an RpS5b mutant background worsened the RpS5b phenotype and blocked oogenesis before egg chambers form. A broad spectrum of mRNAs co-purified in immunoprecipitations with RpS5a, while RpS5b-associated mRNAs were specifically enriched for GO terms related to mitochondrial electron transport and cellular metabolic processes. Consistent with this, RpS5b mitochondrial fractions are depleted for proteins linked to oxidative phosphorylation and mitochondrial respiration, and RpS5b mitochondria tended to form large clusters and had more heterogeneous morphology than those from controls. We conclude that RpS5b-containing ribosomes preferentially associate with particular mRNAs and serve an essential function in oogenesis.
Assuntos
Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Oogênese/genética , Isoformas de Proteínas/genética , RNA/genética , Proteínas Ribossômicas/genética , Ribossomos/genética , Animais , Animais Geneticamente Modificados/genética , Apoptose/genética , Feminino , Células Germinativas/fisiologia , Mitocôndrias/genética , Oócitos/fisiologia , Ovário/fisiologiaRESUMO
The data presented in this article is related to the research article entitled "Biochemical Fractionation of Time-Resolved Drosophila Embryos Reveals Similar Transcriptomic Alterations in Replication Checkpoint and Histone mRNA Processing Mutants" (Lefebvre et al., 2017) [1]. This article provides a spatiotemporal transcriptomic analysis of early embryogenesis and shows that mutations in the checkpoint factor grapes/Chk1 and the histone mRNA processing factor SLBP selectively impair zygotic gene expression. Here, lists of transcripts enriched in early syncytial embryos, late blastoderm embryos, cytoplasmic and nuclear extracts of blastoderm embryos are made public, along with transcription factor motif occurrence for genes enriched in each context. In addition, extensive lists of genes down-regulated upon Chk1 and SLBP protein depletion in embryos are released to enable further analyses.