RESUMEN
Two isoforms of the nuclear pore complex (NPC) have been identified in the yeast S. cerevisiae, which coexist at the periphery of the nucleus and differ by the presence or absence of a nuclear basket. Here, we present a protocol to isolate the two types of NPCs from the same cell extract and dissect their interactomes. We describe steps for powder preparation and magnetic bead conjunction and detail differential affinity purification and outcome evaluation through SDS-PAGE, silver staining, and mass spectrometry analysis. For complete details on the use and execution of this protocol, please refer to Bensidoun et al.1.
Asunto(s)
Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Saccharomyces cerevisiae/metabolismo , Poro Nuclear/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Isoformas de Proteínas/metabolismo , Espectrometría de MasasRESUMEN
To determine which transcripts should reach the cytoplasm for translation, eukaryotic cells have established mechanisms to regulate selective mRNA export through the nuclear pore complex (NPC). The nuclear basket, a substructure of the NPC protruding into the nucleoplasm, is thought to function as a stable platform where mRNA-protein complexes (mRNPs) are rearranged and undergo quality control prior to export, ensuring that only mature mRNAs reach the cytoplasm. Here, we use proteomic, genetic, live-cell, and single-molecule resolution microscopy approaches in budding yeast to demonstrate that basket formation is dependent on RNA polymerase II transcription and subsequent mRNP processing. We further show that while all NPCs can bind Mlp1, baskets assemble only on a subset of nucleoplasmic NPCs, and these basket-containing NPCs associate a distinct protein and RNA interactome. Taken together, our data point toward NPC heterogeneity and an RNA-dependent mechanism for functionalization of NPCs in budding yeast through nuclear basket assembly.
Asunto(s)
Poro Nuclear , Saccharomycetales , Poro Nuclear/genética , Poro Nuclear/metabolismo , Saccharomycetales/genética , Saccharomycetales/metabolismo , ARN Polimerasa II/genética , ARN Polimerasa II/metabolismo , Proteómica , Transporte Activo de Núcleo Celular/fisiología , Núcleo Celular/genética , Núcleo Celular/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Proteínas de Complejo Poro Nuclear/genética , Proteínas de Complejo Poro Nuclear/metabolismoRESUMEN
The preconceptual, intrauterine, and early life environments can have a profound and long-lasting impact on the developmental trajectories and health outcomes of the offspring. Given the relatively low success rates of assisted reproductive technologies (ART; â¼25%), additives and adjuvants, such as glucocorticoids, are used to improve the success rate. Considering the dynamic developmental events that occur during this window, these exposures may alter blastocyst formation at a molecular level, and as such, affect not only the viability of the embryo and the ability of the blastocyst to implant, but also the developmental trajectory of the first three cell lineages, ultimately influencing the physiology of the embryo. In this study, we present a comprehensive single-cell transcriptome, methylome, and small RNA atlas in the day 7 human embryo. We show that, despite no change in morphology and developmental features, preimplantation glucocorticoid exposure reprograms the molecular profile of the TE lineage, and these changes are associated with an altered metabolic and inflammatory response. Our data also suggest that glucocorticoids can precociously mature the TE sublineages, supported by the presence of extravillous trophoblast markers in the polar sublineage and presence of X Chromosome dosage compensation. Further, we have elucidated that epigenetic regulation-DNA methylation and microRNAs (miRNAs)-likely underlies the transcriptional changes observed. This study suggests that exposures to exogenous compounds during preimplantation may unintentionally reprogram the human embryo, possibly leading to suboptimal development and longer-term health outcomes.
RESUMEN
The nuclear pore complex (NPC) serves as a central gate for mRNAs to transit from the nucleus to the cytoplasm. The ability for mRNAs to get exported is linked to various upstream nuclear processes including co-transcriptional RNP assembly and processing, and only export competent mRNPs are thought to get access to the NPC. While the nuclear pore is generally viewed as a monolithic structure that serves as a mediator of transport driven by transport receptors, more recent evidence suggests that the NPC might be more heterogenous than previously believed, both in its composition or in the selective treatment of cargo that seek access to the pore, providing functional plasticity to mRNA export. In this review, we consider the interconnected processes of nuclear mRNA metabolism that contribute and mediate export competence. Furthermore, we examine different aspects of NPC heterogeneity, including the role of the nuclear basket and its associated complexes in regulating selective and/or efficient binding to and transport through the pore. This article is categorized under: RNA Export and Localization > Nuclear Export/Import RNA Turnover and Surveillance > Turnover/Surveillance Mechanisms RNA Interactions with Proteins and Other Molecules > Protein-RNA Interactions: Functional Implications.
Asunto(s)
Poro Nuclear , Transporte de ARN , Transporte Activo de Núcleo Celular , Núcleo Celular/metabolismo , Poro Nuclear/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismoRESUMEN
While biomolecular condensates have emerged as an important biological phenomenon, mechanisms regulating their composition and the ways that viruses hijack these mechanisms remain unclear. The mosquito-borne alphaviruses cause a range of diseases from rashes and arthritis to encephalitis, and no licensed drugs are available for treatment or vaccines for prevention. The alphavirus virulence factor nonstructural protein 3 (nsP3) suppresses the formation of stress granules (SGs)-a class of cytoplasmic condensates enriched with translation initiation factors and formed during the early stage of infection. nsP3 has a conserved N-terminal macrodomain that hydrolyzes ADP-ribose from ADP-ribosylated proteins and a C-terminal hypervariable domain that binds the essential SG component G3BP1. Here, we show that macrodomain hydrolase activity reduces the ADP-ribosylation of G3BP1, disassembles virus-induced SGs, and suppresses SG formation. Expression of nsP3 results in the formation of a distinct class of condensates that lack translation initiation factors but contain G3BP1 and other SG-associated RNA-binding proteins. Expression of ADP-ribosylhydrolase-deficient nsP3 results in condensates that retain translation initiation factors as well as RNA-binding proteins, similar to SGs. Therefore, our data reveal that ADP-ribosylation controls the composition of biomolecular condensates, specifically the localization of translation initiation factors, during alphavirus infection.
Asunto(s)
Alphavirus/genética , ADN Helicasas/genética , N-Glicosil Hidrolasas/genética , Proteínas de Unión a Poli-ADP-Ribosa/genética , ARN Helicasas/genética , Proteínas con Motivos de Reconocimiento de ARN/genética , Proteínas no Estructurales Virales/genética , Alphavirus/patogenicidad , Animales , Artritis/virología , Culicidae/virología , Encefalitis/virología , Exantema/virología , Regulación Viral de la Expresión Génica/genética , Células HeLa , Humanos , Proteínas de Unión al ARN/genéticaRESUMEN
mRNAs and lncRNAs assemble with RNA-binding proteins (RBPs) to form ribonucleoprotein complexes (RNPs ). The assembly of RNPs initiates co-transcriptionally, and their composition and organization is thought to change during the different steps of an RNP life cycle. Modulation of RNP structural organization has been implicated in the regulation of different aspects of RNA metabolism, including establishing interactions between the 5' and 3' ends in regulating mRNA translation and turnover. In this chapter, we describe a single-molecule microscopy approach that combines fluorescent RNA in situ hybridization (smFISH) and structured illumination microscopy (SIM ) and allows to measure different aspects of RNP organization in cells, including distances between different regions within individual mRNAs, as well as the overall compaction state of RNAs in different subcellular compartments and environmental conditions. Moreover, we describe a detailed workflow required for image registration and analysis that allows determining distances at sub-diffraction resolution.
Asunto(s)
Hibridación Fluorescente in Situ/métodos , ARN Largo no Codificante/química , ARN Mensajero/química , Proteínas de Unión al ARN/química , Ribonucleoproteínas/química , Imagen Individual de Molécula/métodos , Conformación de Ácido NucleicoRESUMEN
Cells are complex assemblies of molecules organized into organelles and membraneless compartments, each playing important roles in ensuring cellular homeostasis. The different steps of the gene expression pathway take place within these various cellular compartments, and studying gene regulation and RNA metabolism requires incorporating the spatial as well as temporal separation and progression of these processes. Microscopy has been a valuable tool to study RNA metabolism, as it allows the study of biomolecules in the context of intact individual cells, embryos or tissues, preserving cellular context often lost in experimental approaches that require the collection and lysis of cells in large numbers to obtain sufficient material for different types of assays. Indeed, from the first detection of RNAs and ribosomes in cells to today's ability to study the behaviour of single RNA molecules in living cells, or the expression profile and localization of hundreds of mRNA simultaneously in cells, constant effort in developing tools for microscopy has extensively contributed to our understanding of gene regulation. In this chapter, we will describe the role various microscopy approaches have played in shaping our current understanding of mRNA metabolism and outline how continuous development of new approaches might help in finding answers to outstanding questions or help to look at old dogmas through a new lens.
Asunto(s)
Precursores del ARN , ARN Mensajero , Animales , Expresión Génica , Humanos , Hibridación Fluorescente in Situ , Imagen Molecular , ARN Mensajero/metabolismoRESUMEN
Single-molecule resolution imaging has become an important tool in the study of cell biology. Aptamer-based approaches (e.g., MS2 and PP7) allow for detection of single RNA molecules in living cells and have been used to study various aspects of mRNA metabolism, including mRNP nuclear export. Here we outline an imaging protocol for the study of interactions between mRNPs and nuclear pore complexes (NPCs) in the yeast S. cerevisiae, including mRNP export. We describe in detail the steps that allow for high-resolution live-cell mRNP imaging and measurement of mRNP interactions with NPCs using simultaneous two-color imaging. Our protocol discusses yeast strain construction, choice of marker proteins to label the nuclear pore complex, as well as imaging conditions that allow high signal-to-noise data acquisition. Moreover, we describe various aspects of postacquisition image analysis for single molecule tracking and image registration allowing for the characterization of mRNP-NPC interactions.
Asunto(s)
Microscopía Fluorescente , Imagen Molecular/métodos , Poro Nuclear/metabolismo , Ribonucleoproteínas/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Imagen Individual de Molécula/métodos , Transporte Activo de Núcleo Celular , Regulación Fúngica de la Expresión Génica , Poro Nuclear/genética , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Ribonucleoproteínas/genética , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Factores de TiempoRESUMEN
mRNAs form ribonucleoprotein complexes (mRNPs) by association with proteins that are crucial for mRNA metabolism. While the mRNP proteome has been well characterized, little is known about mRNP organization. Using a single-molecule approach, we show that mRNA conformation changes depending on its cellular localization and translational state. Compared to nuclear mRNPs and lncRNPs, association with ribosomes decompacts individual mRNAs, while pharmacologically dissociating ribosomes or sequestering them into stress granules leads to increased compaction. Moreover, translating mRNAs rarely show co-localized 5' and 3' ends, indicating either that mRNAs are not translated in a closed-loop configuration, or that mRNA circularization is transient, suggesting that a stable closed-loop conformation is not a universal state for all translating mRNAs.
Asunto(s)
Precursores del ARN/fisiología , Ribonucleoproteínas/genética , Ribonucleoproteínas/fisiología , Exones , Expresión Génica/fisiología , Células HEK293 , Humanos , Biosíntesis de Proteínas/fisiología , Precursores del ARN/genética , Empalme del ARN , Estabilidad del ARN , ARN Largo no Codificante , ARN Mensajero/genética , ARN Mensajero/ultraestructura , Ribosomas , Imagen Individual de Molécula/métodos , Análisis EspacialRESUMEN
Cellular senescence is a tumour suppressor programme characterized by a stable cell cycle arrest. Here we report that cellular senescence triggered by a variety of stimuli leads to diminished ribosome biogenesis and the accumulation of both rRNA precursors and ribosomal proteins. These defects were associated with reduced expression of several ribosome biogenesis factors, the knockdown of which was also sufficient to induce senescence. Genetic analysis revealed that Rb but not p53 was required for the senescence response to altered ribosome biogenesis. Mechanistically, the ribosomal protein S14 (RPS14 or uS11) accumulates in the soluble non-ribosomal fraction of senescent cells, where it binds and inhibits CDK4 (cyclin-dependent kinase 4). Overexpression of RPS14 is sufficient to inhibit Rb phosphorylation, inducing cell cycle arrest and senescence. Here we describe a mechanism for maintaining the senescent cell cycle arrest that may be relevant for cancer therapy, as well as biomarkers to identify senescent cells.
Asunto(s)
Puntos de Control del Ciclo Celular , Senescencia Celular , Neoplasias/metabolismo , Proteína de Retinoblastoma/metabolismo , Ribosomas/metabolismo , Factores de Coagulación Sanguínea/genética , Factores de Coagulación Sanguínea/metabolismo , Quinasa 4 Dependiente de la Ciclina/genética , Quinasa 4 Dependiente de la Ciclina/metabolismo , Células HEK293 , Humanos , Neoplasias/genética , Neoplasias/patología , Células PC-3 , Fosforilación , Unión Proteica , Precursores del ARN/biosíntesis , Precursores del ARN/genética , ARN Ribosómico/biosíntesis , ARN Ribosómico/genética , Proteínas de Unión al ARN , Proteína de Retinoblastoma/genética , Proteínas Ribosómicas/genética , Proteínas Ribosómicas/metabolismo , Ribosomas/genética , Transducción de Señal , Factores de TiempoRESUMEN
Lymphocytic choriomeningitis mammarenavirus (LCMV) is an enveloped, negative-strand RNA virus that causes serious disease in humans but establishes an asymptomatic, lifelong infection in reservoir rodents. Different models have been proposed to describe how arenaviruses regulate the replication and transcription of their bisegmented, single-stranded RNA genomes, particularly during persistent infection. However, these models were based largely on viral RNA profiling data derived from entire populations of cells. To better understand LCMV replication and transcription at the single-cell level, we established a high-throughput, single-molecule fluorescence in situ hybridization (smFISH) image acquisition and analysis pipeline and examined viral RNA species at discrete time points from virus entry through the late stages of persistent infection in vitro We observed the transcription of viral nucleoprotein and polymerase mRNAs from the incoming S and L segment genomic RNAs, respectively, within 1 h of infection, whereas the transcription of glycoprotein mRNA from the S segment antigenome required â¼4 to 6 h. This confirms the temporal separation of viral gene expression expected due to the ambisense coding strategy of arenaviruses and also suggests that antigenomic RNA contained in virions is not transcriptionally active upon entry. Viral replication and transcription peaked at 36 h postinfection, followed by a progressive loss of viral RNAs over the next several days. During persistence, the majority of cells showed repeating cyclical waves of viral transcription and replication followed by the clearance of viral RNA. Thus, our data support a model of LCMV persistence whereby infected cells can spontaneously clear infection and become reinfected by viral reservoir cells that remain in the population.IMPORTANCE Arenaviruses are human pathogens that can establish asymptomatic, lifelong infections in their rodent reservoirs. Several models have been proposed to explain how arenavirus spread is restricted within host rodents, including the periodic accumulation and loss of replication-competent, but transcriptionally incompetent, viral genomes. A limitation of previous studies was the inability to enumerate viral RNA species at the single-cell level. We developed a high-throughput, smFISH assay and used it to quantitate lymphocytic choriomeningitis mammarenavirus (LCMV) replicative and transcriptional RNA species in individual cells at distinct time points following infection. Our findings support a model whereby productively infected cells can clear infection, including viral RNAs and antigen, and later be reinfected. This information improves our understanding of the timing and possible regulation of LCMV genome replication and transcription during infection. Importantly, the smFISH assay and data analysis pipeline developed here is easily adaptable to other RNA viruses.
Asunto(s)
Hibridación Fluorescente in Situ/métodos , Virus de la Coriomeningitis Linfocítica/genética , ARN Viral/genética , Células A549 , Línea Celular , Genoma Viral/genética , Humanos , Sondas ARN/genética , Coloración y Etiquetado/métodos , Replicación Viral/genéticaRESUMEN
Cellular mRNA levels are determined by the rates of mRNA synthesis and mRNA decay. Typically, mRNA degradation kinetics are measured on a population of cells that are either chemically treated or genetically engineered to inhibit transcription. However, these manipulations can affect the mRNA decay process itself by inhibiting regulatory mechanisms that govern mRNA degradation, especially if they occur on short time-scales. Recently, single molecule fluorescent in situ hybridization (smFISH) approaches have been implemented to quantify mRNA decay rates in single, unperturbed cells. Here, we provide a step-by-step protocol that allows quantification of mRNA decay in single Saccharomyces cerevisiae using smFISH. Our approach relies on fluorescent labeling of single cytoplasmic mRNAs and nascent mRNAs found at active sites of transcription, coupled with mathematical modeling to derive mRNA half-lives. Commercially available, single-stranded smFISH DNA oligonucleotides (smFISH probes) are used to fluorescently label mRNAs followed by the quantification of cellular and nascent mRNAs using freely available spot detection algorithms. Our method enables quantification of mRNA decay of any mRNA in single, unperturbed yeast cells and can be implemented to quantify mRNA turnover in a variety of cell types as well as tissues.
Asunto(s)
Hibridación Fluorescente in Situ/métodos , Estabilidad del ARN , ARN Mensajero/química , Saccharomyces cerevisiae/química , Análisis de la Célula Individual/métodos , Algoritmos , Citoplasma/química , Citoplasma/genética , Cinética , Modelos Biológicos , Saccharomyces cerevisiae/genética , Transcripción GenéticaRESUMEN
To counteract the breakdown of genome integrity, eukaryotic cells have developed a network of surveillance pathways to prevent and resolve DNA damage. Recent data has recognized the importance of RNA binding proteins (RBPs) in DNA damage repair (DDR) pathways. Here, we describe Nol12 as a multifunctional RBP with roles in RNA metabolism and genome maintenance. Nol12 is found in different subcellular compartments-nucleoli, where it associates with ribosomal RNA and is required for efficient separation of large and small subunit precursors at site 2; the nucleoplasm, where it co-localizes with the RNA/DNA helicase Dhx9 and paraspeckles; as well as GW/P-bodies in the cytoplasm. Loss of Nol12 results in the inability of cells to recover from DNA stress and a rapid p53-independent ATR-Chk1-mediated apoptotic response. Nol12 co-localizes with DNA repair proteins in vivo including Dhx9, as well as with TOPBP1 at sites of replication stalls, suggesting a role for Nol12 in the resolution of DNA stress and maintenance of genome integrity. Identification of a complex Nol12 interactome, which includes NONO, Dhx9, DNA-PK and Stau1, further supports the protein's diverse functions in RNA metabolism and DNA maintenance, establishing Nol12 as a multifunctional RBP essential for genome integrity.
Asunto(s)
ADN/metabolismo , Proteínas Nucleares/metabolismo , ARN Ribosómico/metabolismo , Proteínas de Unión al ARN/metabolismo , Apoptosis , Proteínas de la Ataxia Telangiectasia Mutada/metabolismo , Puntos de Control del Ciclo Celular , Línea Celular , Reparación del ADN , Humanos , Proteínas Nucleares/química , Dominios Proteicos , Proteínas de Unión al ARN/químicaRESUMEN
We report a fluorescence in situ hybridization (FISH) assay that allows the visualization of lymphocytic choriomeningitis mammarenavirus (LCMV) genomic RNAs in individual cells. We show that viral S segment genomic and antigenomic RNA, along with viral nucleoprotein, colocalize in subcellular structures we presume to be viral replication factories. These viral RNA structures are highly dynamic during acute infection, with the many small foci seen early coalescing into larger perinuclear foci later in infection. These late-forming perinuclear viral RNA aggregates are located near the cellular microtubule organizing centre and colocalize with the early endosomal marker Rab5c and the viral glycoprotein in a proportion of infected cells. We propose that the virus is using the surface of a cellular membrane-bound organelle as a site for the pre-assembly of viral components, including genomic RNA and viral glycoprotein, prior to their transport to the plasma membrane, where new particles will bud.
RESUMEN
Enhancers are intergenic DNA elements that regulate the transcription of target genes in response to signaling pathways by interacting with promoters over large genomic distances. Recent studies have revealed that enhancers are bi-directionally transcribed into enhancer RNAs (eRNAs). Using single-molecule fluorescence in situ hybridization (smFISH), we investigated the eRNA-mediated regulation of transcription during estrogen induction in MCF-7 cells. We demonstrate that eRNAs are localized exclusively in the nucleus and are induced with similar kinetics as target mRNAs. However, eRNAs are mostly nascent at enhancers and their steady-state levels remain lower than those of their cognate mRNAs. Surprisingly, at the single-allele level, eRNAs are rarely co-expressed with their target loci, demonstrating that active gene transcription does not require the continuous transcription of eRNAs or their accumulation at enhancers. When co-expressed, sub-diffraction distance measurements between nascent mRNA and eRNA signals reveal that co-transcription of eRNAs and mRNAs rarely occurs within closed enhancer-promoter loops. Lastly, basal eRNA transcription at enhancers, but not E2-induced transcription, is maintained upon depletion of MLL1 and ERα, suggesting some degree of chromatin accessibility prior to signal-dependent activation of transcription. Together, our findings suggest that eRNA accumulation at enhancer-promoter loops is not required to sustain target gene transcription.
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Elementos de Facilitación Genéticos , Regiones Promotoras Genéticas , ARN no Traducido/biosíntesis , Transcripción Genética , Estradiol/farmacología , Receptor alfa de Estrógeno/fisiología , Factores de Transcripción Forkhead/biosíntesis , Factores de Transcripción Forkhead/genética , N-Metiltransferasa de Histona-Lisina/fisiología , Humanos , Células MCF-7 , Modelos Moleculares , Proteína de la Leucemia Mieloide-Linfoide/fisiología , ARN Mensajero/biosíntesis , ARN no Traducido/fisiología , Receptores Purinérgicos P2Y2/biosíntesis , Receptores Purinérgicos P2Y2/genética , Análisis de la Célula IndividualRESUMEN
The last steps in mRNA export and remodeling are performed by the Nup82 complex, a large conserved assembly at the cytoplasmic face of the nuclear pore complex (NPC). By integrating diverse structural data, we have determined the molecular architecture of the native Nup82 complex at subnanometer precision. The complex consists of two compositionally identical multiprotein subunits that adopt different configurations. The Nup82 complex fits into the NPC through the outer ring Nup84 complex. Our map shows that this entire 14-MDa Nup82-Nup84 complex assembly positions the cytoplasmic mRNA export factor docking sites and messenger ribonucleoprotein (mRNP) remodeling machinery right over the NPC's central channel rather than on distal cytoplasmic filaments, as previously supposed. We suggest that this configuration efficiently captures and remodels exporting mRNP particles immediately upon reaching the cytoplasmic side of the NPC.
Asunto(s)
Proteínas de Complejo Poro Nuclear/química , Poro Nuclear/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Levaduras/metabolismo , Transporte Activo de Núcleo Celular , Proteínas Fúngicas , Proteínas de Complejo Poro Nuclear/ultraestructura , ARN Mensajero , Saccharomyces cerevisiae/citología , Proteínas de Saccharomyces cerevisiae/ultraestructuraRESUMEN
Regulation of mRNA and protein expression occurs at many levels, initiated at transcription and followed by mRNA processing, export, localization, translation and mRNA degradation. The ability to study mRNAs in living cells has become a critical tool to study and analyze how the various steps of the gene expression pathway are carried out. Here we describe a detailed protocol for real time fluorescent RNA imaging using the PP7 bacteriophage coat protein, which allows mRNA detection with high spatial and temporal resolution in the yeast Saccharomyces cerevisiae, and can be applied to study various stages of mRNA metabolism. We describe the different parameters required for quantitative single molecule imaging in yeast, including strategies for genomic integration, expression of a PP7 coat protein GFP fusion protein, microscope setup and analysis strategies. We illustrate the method's use by analyzing the behavior of nuclear mRNA in yeast and the role of the nuclear basket in mRNA export.
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Regulación Fúngica de la Expresión Génica , ARN de Hongos/química , ARN Mensajero/química , Saccharomyces cerevisiae/ultraestructura , Imagen Individual de Molécula/métodos , Coloración y Etiquetado/métodos , Transporte Activo de Núcleo Celular , Proteínas de la Cápside/genética , Proteínas de la Cápside/metabolismo , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Microscopía Fluorescente/métodos , Biosíntesis de Proteínas , Estabilidad del ARN , Transporte de ARN , ARN de Hongos/genética , ARN de Hongos/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Transcripción GenéticaRESUMEN
Many messenger RNA export proteins have been identified; yet the spatial and temporal activities of these proteins and how they determine directionality of messenger ribonucleoprotein (mRNP) complex export from the nucleus remain largely undefined. Here, the bacteriophage PP7 RNA-labeling system was used in Saccharomyces cerevisiae to follow single-particle mRNP export events with high spatial precision and temporal resolution. These data reveal that mRNP export, consisting of nuclear docking, transport, and cytoplasmic release from a nuclear pore complex (NPC), is fast (â¼ 200 ms) and that upon arrival in the cytoplasm, mRNPs are frequently confined near the nuclear envelope. Mex67p functions as the principal mRNP export receptor in budding yeast. In a mex67-5 mutant, delayed cytoplasmic release from NPCs and retrograde transport of mRNPs was observed. This proves an essential role for Mex67p in cytoplasmic mRNP release and directionality of transport.
Asunto(s)
Núcleo Celular/genética , Núcleo Celular/metabolismo , Proteínas Nucleares/metabolismo , Proteínas de Transporte Nucleocitoplasmático/metabolismo , Transporte de ARN , ARN Mensajero/metabolismo , Proteínas de Unión al ARN/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , ARN de Hongos/metabolismoRESUMEN
After synthesis and transit through the nucleus, messenger RNAs (mRNAs) are exported to the cytoplasm through the nuclear pore complex (NPC). At the NPC, messenger ribonucleoproteins (mRNPs) first encounter the nuclear basket where mRNP rearrangements are thought to allow access to the transport channel. Here, we use single mRNA resolution live cell microscopy and subdiffraction particle tracking to follow individual mRNAs on their path toward the cytoplasm. We show that when reaching the nuclear periphery, RNAs are not immediately exported but scan along the nuclear periphery, likely to find a nuclear pore allowing export. Deletion or mutation of the nuclear basket proteins MLP1/2 or the mRNA binding protein Nab2 changes the scanning behavior of mRNPs at the nuclear periphery, shortens residency time at nuclear pores, and results in frequent release of mRNAs back into the nucleoplasm. These observations suggest a role for the nuclear basket in providing an interaction platform that keeps RNAs at the periphery, possibly to allow mRNP rearrangements before export.
Asunto(s)
Núcleo Celular/genética , Núcleo Celular/metabolismo , ARN Mensajero/metabolismo , Saccharomyces cerevisiae/citología , Saccharomyces cerevisiae/genética , Citoplasma/genética , Citoplasma/metabolismo , Poro Nuclear/metabolismo , Transporte de ARNRESUMEN
Transcription is a highly regulated biological process, initiated through the assembly of complexes at the promoter that contain both the general transcriptional machinery and promoter-specific factors. Despite the abundance of studies focusing on transcription, certain questions have remained unanswered. It is not clear how the transcriptional profile of a promoter is affected by genomic context. Also, there is no single cell method to directly compare transcriptional profiles independent of gene length and sequence. In this work, we employ a single genetic site for isolating the transcriptional kinetics of yeast promoters. Utilizing single molecule FISH, we directly compare the transcriptional activity of different promoters, considering both synthesis and cell-to-cell variability. With this approach, we provide evidence suggesting promoters autonomously encode their associated transcriptional profiles, independent of genomic locus, gene length and gene sequence.