RESUMEN
RNA-binding proteins are key regulators of gene expression, yet only a small fraction have been functionally characterized. Here we report a systematic analysis of the RNA motifs recognized by RNA-binding proteins, encompassing 205 distinct genes from 24 diverse eukaryotes. The sequence specificities of RNA-binding proteins display deep evolutionary conservation, and the recognition preferences for a large fraction of metazoan RNA-binding proteins can thus be inferred from their RNA-binding domain sequence. The motifs that we identify in vitro correlate well with in vivo RNA-binding data. Moreover, we can associate them with distinct functional roles in diverse types of post-transcriptional regulation, enabling new insights into the functions of RNA-binding proteins both in normal physiology and in human disease. These data provide an unprecedented overview of RNA-binding proteins and their targets, and constitute an invaluable resource for determining post-transcriptional regulatory mechanisms in eukaryotes.
Asunto(s)
Regulación de la Expresión Génica/genética , Motivos de Nucleótidos/genética , Proteínas de Unión al ARN/metabolismo , Trastorno Autístico/genética , Secuencia de Bases , Sitios de Unión/genética , Secuencia Conservada/genética , Células Eucariotas/metabolismo , Humanos , Datos de Secuencia Molecular , Estructura Terciaria de Proteína/genética , Factores de Empalme de ARN , Estabilidad del ARN/genética , Proteínas de Unión al ARN/química , Proteínas de Unión al ARN/genéticaRESUMEN
Animal mRNAs are regulated by hundreds of RNA binding proteins (RBPs). The identification of RBP targets is crucial for understanding their function. A recent method, PAR-CLIP, uses photoreactive nucleosides to crosslink RBPs to target RNAs in cells prior to immunoprecipitation. Here, we establish iPAR-CLIP (in vivo PAR-CLIP) to determine, at nucleotide resolution, transcriptome-wide binding sites of GLD-1, a conserved, germline-specific translational repressor in C. elegans. We identified 439 reproducible target mRNAs and demonstrate an excellent dynamic range of target detection by iPAR-CLIP. Upon GLD-1 knockdown, protein but not mRNA expression of the 439 targets was specifically upregulated, demonstrating functionality. Finally, we discovered strongly conserved GLD-1 binding sites near the start codon of target genes. These sites are functional in vitro and likely confer strong repression in vivo. We propose that GLD-1 interacts with the translation machinery near the start codon, a so-far-unknown mode of gene regulation in eukaryotes.
Asunto(s)
Caenorhabditis elegans/genética , ARN de Helminto/genética , ARN de Helminto/metabolismo , Transcriptoma/genética , Animales , Sitios de Unión , Biología Computacional , Reactivos de Enlaces Cruzados/química , Reactivos de Enlaces Cruzados/metabolismo , Inmunoprecipitación , ARN Mensajero/genética , ARN Mensajero/metabolismo , Proteínas de Unión al ARN/metabolismoRESUMEN
Studying protein interactions in whole organisms is fundamental to understanding development. Here, we combine in vivo expressed GFP-tagged proteins with quantitative proteomics to identify protein-protein interactions of selected key proteins involved in early C. elegans embryogenesis. Co-affinity purification of interaction partners for eight bait proteins resulted in a pilot in vivo interaction map of proteins with a focus on early development. Our network reflects known biology and is highly enriched in functionally relevant interactions. To demonstrate the utility of the map, we looked for new regulators of P granule dynamics and found that GEI-12, a novel binding partner of the DYRK family kinase MBK-2, is a key regulator of P granule formation and germline maintenance. Our data corroborate a recently proposed model in which the phosphorylation state of GEI-12 controls P granule dynamics. In addition, we find that GEI-12 also induces granule formation in mammalian cells, suggesting a common regulatory mechanism in worms and humans. Our results show that in vivo interaction proteomics provides unique insights into animal development.
Asunto(s)
Proteínas de Caenorhabditis elegans/análisis , Caenorhabditis elegans/embriología , Proteínas Portadoras/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Tirosina Quinasas/metabolismo , Proteómica/métodos , Animales , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/metabolismo , Cromatografía de Afinidad , Gránulos Citoplasmáticos/metabolismo , Regulación del Desarrollo de la Expresión Génica , Espectrometría de Masas , Fosforilación , Mapas de Interacción de Proteínas , Quinasas DyrKRESUMEN
Hypoxia is a common feature of many solid tumors due to aberrant proliferation and angiogenesis that is associated with tumor progression and metastasis. Most of the well-known hypoxia effects are mediated through hypoxia-inducible factors (HIFs). Identification of the long-lasting effects of hypoxia beyond the immediate HIF-induced alterations could provide a better understanding of hypoxia-driven metastasis and potential strategies to circumvent it. Here, we uncovered a hypoxia-induced mechanism that exerts a prolonged effect to promote metastasis. In breast cancer patient-derived circulating tumor cell (CTC) lines and common breast cancer cell lines, hypoxia downregulated tumor intrinsic type I interferon (IFN) signaling and its downstream antigen presentation (AP) machinery in luminal breast cancer cells, via both HIF-dependent and HIF-independent mechanisms. Hypoxia induced durable IFN/AP suppression in certain cell types that was sustained after returning to normoxic conditions, presenting a "hypoxic memory" phenotype. Hypoxic memory of IFN/AP downregulation was established by specific hypoxic priming, and cells with hypoxic memory had an enhanced ability for tumorigenesis and metastasis. Overexpression of IRF3 enhanced IFN signaling and reduced tumor growth in normoxic, but not hypoxic, conditions. The histone deacetylase inhibitor (HDACi) entinostat upregulated IFN targets and erased the hypoxic memory. These results point to a mechanism by which hypoxia facilitates tumor progression through a long-lasting memory that provides advantages for CTCs during the metastatic cascade.
RESUMEN
BACKGROUND: The 3' untranslated regions (UTRs) of mRNAs play a major role in post-transcriptional regulation of gene expression. Selection of transcript cleavage and polyadenylation sites is a dynamic process that produces multiple transcript isoforms for the same gene within and across different cell types. Using LITE-Seq, a new quantitative method to capture transcript 3' ends expressed in vivo, we have characterized sex- and cell type-specific transcriptome-wide changes in gene expression and 3'UTR diversity in Caenorhabditis elegans germline cells undergoing proliferation and differentiation. RESULTS: We show that nearly half of germline transcripts are alternatively polyadenylated, that differential regulation of endogenous 3'UTR variants is common, and that alternative isoforms direct distinct spatiotemporal protein expression patterns in vivo. Dynamic expression profiling also reveals temporal regulation of X-linked gene expression, selective stabilization of transcripts, and strong evidence for a novel developmental program that promotes nucleolar dissolution in oocytes. We show that the RNA-binding protein NCL-1/Brat is a posttranscriptional regulator of numerous ribosome-related transcripts that acts through specific U-rich binding motifs to down-regulate mRNAs encoding ribosomal protein subunits, rRNA processing factors, and tRNA synthetases. CONCLUSIONS: These results highlight the pervasive nature and functional potential of patterned gene and isoform expression during early animal development.