RESUMEN
The four-dimensional nucleome (4DN) consortium studies the architecture of the genome and the nucleus in space and time. We summarize progress by the consortium and highlight the development of technologies for (1) mapping genome folding and identifying roles of nuclear components and bodies, proteins, and RNA, (2) characterizing nuclear organization with time or single-cell resolution, and (3) imaging of nuclear organization. With these tools, the consortium has provided over 2,000 public datasets. Integrative computational models based on these data are starting to reveal connections between genome structure and function. We then present a forward-looking perspective and outline current aims to (1) delineate dynamics of nuclear architecture at different timescales, from minutes to weeks as cells differentiate, in populations and in single cells, (2) characterize cis-determinants and trans-modulators of genome organization, (3) test functional consequences of changes in cis- and trans-regulators, and (4) develop predictive models of genome structure and function.
Asunto(s)
Núcleo Celular , Genoma , Genoma/genética , Núcleo Celular/genética , Núcleo Celular/metabolismo , Cromatina/metabolismoRESUMEN
How the splicing machinery defines exons or introns as the spliced unit has remained a puzzle for 30 years. Here, we demonstrate that peripheral and central regions of the nucleus harbor genes with two distinct exon-intron GC content architectures that differ in the splicing outcome. Genes with low GC content exons, flanked by long introns with lower GC content, are localized in the periphery, and the exons are defined as the spliced unit. Alternative splicing of these genes results in exon skipping. In contrast, the nuclear center contains genes with a high GC content in the exons and short flanking introns. Most splicing of these genes occurs via intron definition, and aberrant splicing leads to intron retention. We demonstrate that the nuclear periphery and center generate different environments for the regulation of alternative splicing and that two sets of splicing factors form discrete regulatory subnetworks for the two gene architectures. Our study connects 3D genome organization and splicing, thus demonstrating that exon and intron definition modes of splicing occur in different nuclear regions.
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Empalme Alternativo , Empalme del ARN , Composición de Base , Exones/genética , Intrones/genéticaRESUMEN
Stress granules (SGs) are cytoplasmic assemblies formed under various stress conditions as a consequence of translation arrest. SGs contain RNA-binding proteins, ribosomal subunits and messenger RNAs (mRNAs). It is well known that mRNAs contribute to SG formation; however, the connection between SG assembly and nuclear processes that involve mRNAs is not well established. Here, we examine the effects of inhibiting mRNA transcription, splicing and export on the assembly of SGs and the related cytoplasmic P body (PB). We demonstrate that inhibition of mRNA transcription, splicing and export reduces the formation of canonical SGs in a eukaryotic initiation factor 2α phosphorylation-independent manner, and alters PB size and quantity. We find that the splicing inhibitor madrasin promotes the assembly of stress-like granules. We show that the addition of synthetic mRNAs directly to the cytoplasm is sufficient for SG assembly, and that the assembly of these SGs requires the activation of stress-associated protein synthesis pathways. Moreover, we show that adding an excess of mRNA to cells that do not have active splicing, and therefore have low levels of cytoplasmic mRNAs, promotes SG formation under stress conditions. These findings emphasize the importance of the cytoplasmic abundance of newly transcribed mRNAs in the assembly of SGs.
Asunto(s)
Núcleo Celular , Gránulos Citoplasmáticos , ARN Mensajero , Humanos , Núcleo Celular/metabolismo , Núcleo Celular/genética , Citoplasma/metabolismo , Gránulos Citoplasmáticos/metabolismo , Factor 2 Eucariótico de Iniciación/metabolismo , Células HeLa , Fosforilación , Empalme del ARN , ARN Mensajero/metabolismo , ARN Mensajero/genética , Gránulos de Estrés/metabolismo , Transcripción Genética , Transporte Activo de Núcleo CelularRESUMEN
Infectious diseases are one of the world's leading causes of morbidity. Their rapid spread emphasizes the need for accurate and fast diagnostic methods for large-scale screening. Here, we describe a robust method for the detection of pathogens based on microscale thermophoresis (MST). The method involves the hybridization of a fluorescently labeled DNA probe to a target RNA and the assessment of thermophoretic migration of the resulting complex in solution within a 2 to 30-time window. We found that the thermophoretic migration of the nucleic acid-based probes is primarily determined by the fluorescent molecule used, rather than the nucleic acid sequence of the probe. Furthermore, a panel of uniformly labeled probes that bind to the same target RNA yields a more responsive detection pattern than a single probe, and moreover, can be used for the detection of specific pathogen variants. In addition, intercalating agents (ICA) can be used to alter migration directionality to improve detection sensitivity and resolving power by several orders of magnitude. We show that this approach can rapidly diagnose viral SARS-CoV2, influenza H1N1, artificial pathogen targets, and bacterial infections. Furthermore, it can be used for anti-microbial resistance testing within 2 h, demonstrating its diagnostic potential for early pathogen detection.
Asunto(s)
Ensayos Analíticos de Alto Rendimiento , Técnicas Microbiológicas , Técnicas de Diagnóstico Molecular , Hibridación de Ácido Nucleico , ARN , Sondas de ADN , Subtipo H1N1 del Virus de la Influenza A/genética , Subtipo H1N1 del Virus de la Influenza A/aislamiento & purificación , Técnicas de Diagnóstico Molecular/métodos , Técnicas de Diagnóstico Molecular/normas , Técnicas Microbiológicas/métodos , Técnicas Microbiológicas/normas , Ensayos Analíticos de Alto Rendimiento/métodos , Ensayos Analíticos de Alto Rendimiento/normas , ARN/análisis , SARS-CoV-2/genética , SARS-CoV-2/aislamiento & purificación , Virosis/diagnóstico , Infecciones Bacterianas/diagnóstico , Línea Celular Tumoral , HumanosRESUMEN
Nuclear speckles are dynamic membraneless bodies located in the cell nucleus. They harbor RNAs and proteins, many of which are splicing factors, that together display complex biophysical properties dictating nuclear speckle formation and maintenance. Although these nuclear bodies were discovered decades ago, only recently has in-depth genomic analysis begun to unravel their essential functions in modulation of gene activity. Major advancements in genomic mapping techniques combined with microscopy approaches have enabled insights into the roles nuclear speckles may play in enhancing gene expression, and how gene positioning to specific nuclear landmarks can regulate gene expression and RNA processing. Some studies have drawn a link between nuclear speckles and disease. Certain maladies either involve nuclear speckles directly or dictate the localization and reorganization of many nuclear speckle factors. This is most striking during viral infection, as viruses alter the entire nuclear architecture and highjack host machinery. As discussed in this Review, nuclear speckles represent a fascinating target of study not only to reveal the links between gene positioning, genome subcompartments and gene activity, but also as a potential target for therapeutics.
Asunto(s)
Cuerpos Nucleares , Motas Nucleares , Biofisica , Núcleo Celular/genética , Expresión GénicaRESUMEN
Stress granules (SGs) can assemble in cancer cells upon chemotoxic stress. Glucocorticoids function during stress responses and are administered with chemotherapies. The roles of glucocorticoids in SG assembly and disassembly pathways are unknown. We examined whether combining glucocorticoids such as cortisone with chemotherapies from the vinca alkaloid family, which dismantle the microtubule network, affects SG assembly and disassembly pathways and influences cell viability in cancer cells and human-derived organoids. Cortisone augmented SG formation when combined with vinorelbine (VRB). Live-cell imaging showed that cortisone increased SG assembly rates but reduced SG clearance rates after stress, by increasing protein residence times within the SGs. Mechanistically, VRB and cortisone signaled through the integrated stress response mediated by eIF2α (also known as EIF2S1), yet induced different kinases, with cortisone activating the GCN2 kinase (also known as EIF2AK4). Cortisone increased VRB-induced cell death and reduced the population of cells trapped in mitotic catastrophe. These effects were mediated by the core SG proteins G3BP1 and G3BP2. In conclusion, glucocorticoids induce SG assembly and cell death when administered with chemotherapies, suggesting that combining glucocorticoids with chemotherapies can enhance cancer cell chemosensitivity.
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Cortisona , Glucocorticoides , Muerte Celular , Cortisona/metabolismo , Gránulos Citoplasmáticos/metabolismo , ADN Helicasas , Glucocorticoides/farmacología , Humanos , Proteínas de Unión a Poli-ADP-Ribosa/metabolismo , Proteínas Serina-Treonina Quinasas , ARN Helicasas/metabolismo , Proteínas con Motivos de Reconocimiento de ARN/metabolismo , Gránulos de EstrésRESUMEN
Super-enhancers are unique gene expression regulators widely involved in cancer development. Spread over large DNA segments, they tend to be found next to oncogenes. The super-enhancer c-MYC locus forms long-range chromatin looping with nearby genes, which brings the enhancer and the genes into proximity, to promote gene activation. The colon cancer-associated transcript 1 (CCAT1) gene, which is part of the MYC locus, transcribes a lncRNA that is overexpressed in colon cancer cells through activation by MYC. Comparing different types of cancer cell lines using RNA fluorescence in situ hybridization (RNA FISH), we detected very prominent CCAT1 expression in HeLa cells, observed as several large CCAT1 nuclear foci. We found that dozens of CCAT1 transcripts accumulate on the gene locus, in addition to active transcription occurring from the gene. The accumulating transcripts are released from the chromatin during cell division. Examination of CCAT1 lncRNA expression patterns on the single-RNA level showed that unspliced CCAT1 transcripts are released from the gene into the nucleoplasm. Most of these unspliced transcripts were observed in proximity to the active gene but were not associated with nuclear speckles in which unspliced RNAs usually accumulate. At larger distances from the gene, the CCAT1 transcripts appeared spliced, implying that most CCAT1 transcripts undergo post-transcriptional splicing in the zone of the active gene. Finally, we show that unspliced CCAT1 transcripts can be detected in the cytoplasm during splicing inhibition, which suggests that there are several CCAT1 variants, spliced and unspliced, that the cell can recognize as suitable for export.
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Cromatina , ARN Largo no Codificante , Humanos , ARN Largo no Codificante/genética , ARN Largo no Codificante/metabolismo , Cromatina/metabolismo , Cromatina/genética , Cromatina/química , Empalme del ARN , Células HeLa , Hibridación Fluorescente in SituRESUMEN
Endogenous gene knock-in using CRIPSR is becoming the standard for fluorescent tagging of endogenous proteins. Some protocols, particularly those that utilize insert cassettes that carry a fluorescent protein tag, can yield many types of cells with off-target insertions that have diffuse fluorescent signal throughout the whole cell in addition to scarce cells with on-target gene insertions that show the correct sub-cellular localization of the tagged protein. As such, when searching for cells with on-target integration using flow cytometry, the off-target fluorescent cells yield a high percentage of false positives. Here, we show that by changing the gating used to select for fluorescence during flow cytometry sorting, namely utilizing the width of the signal as opposed to the area, we can highly enrich for positively integrated cells. Reproducible gates were created to select even minuscule percentages of correct subcellular signal, and these parameters were validated by fluorescence microscopy. This method is a powerful tool to rapidly enhance the generation of cell lines with correctly integrated gene knock-ins encoding endogenous fluorescent proteins.
Asunto(s)
Colorantes , Proteínas , Citometría de Flujo , Línea Celular , Microscopía FluorescenteRESUMEN
Nuclear speckles are eukaryotic nuclear bodies enriched in splicing factors. Their exact purpose has been a matter of debate. The different proposed roles of nuclear speckles are reviewed and an additional layer of function is put forward, suggesting that by accumulating splicing factors within them, nuclear speckles can buffer the nucleoplasmic levels of splicing factors available for splicing and thereby modulate splicing rates. These findings build on the already established model that nuclear speckles function as a storage/recycling site for splicing factors. Many studies have demonstrated proximity between nuclear speckles and sites of active transcription, suggesting that this juxtaposition can enhance the rates of gene expression. It is found that nuclear speckle disassembly increases splicing factor availability in the nucleoplasm, leading to an increase in splicing rates and faster release of nascent transcripts from the gene after transcription. Altogether, this era in which genomic and imaging approaches are applied to study nuclear organization has expanded the outlook on the possible roles of nuclear speckles.
Asunto(s)
Núcleo Celular , ARN , Núcleo Celular/genética , Núcleo Celular/metabolismo , Expresión Génica , Células HeLa , Humanos , ARN/metabolismo , Empalme del ARN/genética , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismoRESUMEN
Gene expression dynamics can be measured in single living cells. Using a detectable transcriptionally active gene in living cells, we previously found that an mRNA undergoing several splicing events was retained at this gene after transcription until completion of mRNA processing. To determine the reason for this delay in release and whether mRNA retention on the gene might depend on splicing factor availability, we modulated the levels of splicing factors in the nucleus. Increasing the abundance of the diffusing fraction of splicing factors by their overexpression or by Clk1 kinase overexpression to disassemble nuclear speckles, led to a reduction in splicing factor residence times on the active gene, and the retained mRNA was rapidly released from the gene. Other treatments such as overexpression of a mutant inactive Clk1, the downregulation of MALAT1 lncRNA or of the Son protein, or the overexpression of the splicing factor import factor TNPO3, did not affect the dynamics of mRNA release from the gene. We found that the faster release of the mRNA from the gene mediated by increased availability of splicing factors, was dependent on the RS domain of the splicing factors and its phosphorylation state. We propose that the relative abundancies of splicing factors in the nucleoplasm can affect their availability for the splicing events taking place, and regulate the kinetics of mRNA release from the gene after processing.
Asunto(s)
Factores de Empalme de ARN/genética , Empalme del ARN/genética , Transcripción Genética , Proteínas de Unión al ADN/genética , Regulación de la Expresión Génica/genética , Células HeLa , Humanos , Intrones/genética , Antígenos de Histocompatibilidad Menor/genética , Fosforilación , Unión Proteica/genética , Proteínas Serina-Treonina Quinasas/genética , Proteínas Tirosina Quinasas/genética , Precursores del ARN/genética , ARN Largo no Codificante/genética , ARN Mensajero/genética , ARN Interferente Pequeño/genética , beta Carioferinas/genéticaRESUMEN
RNA editing results in the site-specific conversion of adenosine to inosine in mRNAs. Genomics has revealed millions of editing sites in metazoans, but examining the spatial aspects of editing in cells has been challenging. A new method, inosineFISH (inoFISH), provides the ability to detect edited and unedited mRNAs within intact cells.
Asunto(s)
Núcleo Celular/genética , Edición de ARN/genética , ARN Mensajero/genética , Animales , Humanos , Hibridación Fluorescente in Situ , Inosina/químicaRESUMEN
The genetic information encoded in nuclear mRNA destined to reach the cytoplasm requires the interaction of the mRNA molecule with the nuclear pore complex (NPC) for the process of mRNA export. Numerous proteins have important roles in the transport of mRNA out of the nucleus. The NPC embedded in the nuclear envelope is the port of exit for mRNA and is composed of â¼30 unique proteins, nucleoporins, forming the distinct structures of the nuclear basket, the pore channel and cytoplasmic filaments. Together, they serve as a rather stationary complex engaged in mRNA export, while a variety of soluble protein factors dynamically assemble on the mRNA and mediate the interactions of the mRNA with the NPC. mRNA export factors are recruited to and dissociate from the mRNA at the site of transcription on the gene, during the journey through the nucleoplasm and at the nuclear pore at the final stages of export. In this review, we present the current knowledge derived from biochemical, molecular, structural and imaging studies, to develop a high-resolution picture of the many events that culminate in the successful passage of the mRNA out of the nucleus.
Asunto(s)
Transporte Activo de Núcleo Celular/fisiología , Proteínas de Complejo Poro Nuclear , Poro Nuclear , Transporte de ARN/fisiología , ARN Mensajero/metabolismo , Animales , Núcleo Celular/metabolismo , Chlamydomonas reinhardtii/citología , Chlamydomonas reinhardtii/metabolismo , Citoplasma/metabolismo , Humanos , Membrana Nuclear/metabolismo , Poro Nuclear/química , Poro Nuclear/metabolismo , Proteínas de Complejo Poro Nuclear/química , Proteínas de Complejo Poro Nuclear/metabolismo , Proteínas Nucleares/metabolismo , ARN Viral/metabolismo , Saccharomyces cerevisiae/citología , Saccharomyces cerevisiae/metabolismoRESUMEN
Fluorescence in situ hybridization (FISH) can be used for the intracellular detection of DNA or RNA molecules. The detection of DNA sequences by DNA FISH requires the denaturation of the DNA double helix to allow the hybridization of the fluorescent probe with DNA in a single stranded form. These hybridization conditions require high temperature and low pH that can damage RNA, and therefore RNA is not typically detectable by DNA FISH. In contrast, RNA FISH does not require a denaturation step since RNA is single stranded, and therefore DNA molecules are not detectable by RNA FISH. Hence, DNA FISH and RNA FISH are mutually exclusive. In this study, we show that plasmid DNA transiently transfected into cells is readily detectable in the cytoplasm by RNA FISH without need for denaturation, shortly after transfection and for several hours. The plasmids, however, are usually not detectable in the nucleus except when the plasmids are efficiently directed into the nucleus, which may imply a more open packaging state for DNA after transfection. This detection of plasmid DNA in the cytoplasm has implications for RNA FISH experiments and opens a window to study conditions when DNA is present in the cytoplasm.
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Citoplasma/ultraestructura , ADN/ultraestructura , Hibridación Fluorescente in Situ/métodos , ARN/química , Núcleo Celular/ultraestructura , ADN/aislamiento & purificación , Colorantes Fluorescentes/química , Hibridación de Ácido Nucleico , Plásmidos/genética , Secuencias Repetitivas de Ácidos NucleicosRESUMEN
Eukaryotic cells contain sub-cellular compartments that are not membrane bound. Some structures are always present, such as nuclear speckles that contain RNA-binding proteins (RBPs) and poly(A)+ RNAs. Others, like cytoplasmic stress granules (SGs) that harbor mRNAs and RBPs, are induced upon stress. When we examined the formation and composition of nuclear speckles during stress induction with tubercidin, an adenosine analogue previously shown to affect nuclear speckle composition, we unexpectedly found that it also led to the formation of SGs and to the inhibition of several crucial steps of RNA metabolism in cells, thereby serving as a potent inhibitor of the gene expression pathway. Although transcription and splicing persisted under this stress, RBPs and mRNAs were mislocalized in the nucleus and cytoplasm. Specifically, lncRNA and RBP localization to nuclear speckles was disrupted, exon junction complex (EJC) recruitment to mRNA was reduced, mRNA export was obstructed, and cytoplasmic poly(A)+ RNAs localized in SGs. Furthermore, nuclear proteins that participate in mRNA export, such as nucleoporins and mRNA export adaptors, were mislocalized to SGs. This study reveals structural aspects of granule assembly in cells, and describes how the flow of RNA from the nucleus to the cytoplasm is severed under stress.
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Proteínas de Complejo Poro Nuclear/genética , Transporte de ARN/genética , ARN Largo no Codificante/genética , ARN/genética , Transporte Activo de Núcleo Celular/genética , Adenosina/química , Adenosina/genética , Núcleo Celular/genética , Núcleo Celular/metabolismo , Citoplasma , Gránulos Citoplasmáticos/genética , Gránulos Citoplasmáticos/metabolismo , Estructuras Citoplasmáticas/genética , Exones/genética , Humanos , Empalme del ARN/genética , ARN Mensajero/genética , Proteínas de Unión al ARN/genética , Estrés Fisiológico/genética , Tubercidina/químicaRESUMEN
Currently, there is demand for fluorescent oligonucleotide probes for diagnostic purposes. To address this necessity, we developed nucleosides containing a flexible spacer with an intercalating moiety at its end (NIC molecules). The intercalator is based on 4-hydroxybenzylidene imidazolinone (HBI), found in the Green Fluorescent Protein. We synthesized 20-mer oligonucleotides, ON1-ON4, incorporating the DMTr phosphorodiamidite monomer of dUHBI, 2, and the corresponding dUDFHBI, 5b, monomer. ON1-ON4 target the HER-2 mRNA breast cancer marker for the diagnostics of breast cancer subtype. Hybridization of ON1/ON2 and ON3/ON4 with complementary 2'-OMe-RNA resulted in emission at 462 and 481 nm, respectively, and up to 46-fold increase in fluorescence intensity. CD and 19F-NMR data indicated that HBI and DFHBI fluorophores bind as intercalators and stabilize the duplexes (up to ΔTm 6 °C). Furthermore, addition of ON1-ON4 to total RNA extracted from cancer cells that overexpress HER-2 mRNA, resulted in a significant fluorescence enhancement of ON3 and ON4. The latter sensitively detected low concentrations of the target mRNA (at total RNA 30 ng/µL). These probes were photostable for 200 min. Using a dilution curve, we quantified the number of HER-2 transcripts in a cell. In conclusion, ON3 and ON4 are promising diagnostic probes for an easy, instantaneous, specific, and sensitive detection of levels of oncogenes. Importantly, the NIC concept, demonstrated here for diagnostics of breast cancer, is universal and may be applied not only in a clinical setting but also for the detection of any RNA.
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Biomarcadores de Tumor/genética , Neoplasias de la Mama/genética , Colorantes Fluorescentes/química , Límite de Detección , Receptor ErbB-2/genética , Línea Celular Tumoral , Humanos , Hibridación de Ácido Nucleico , ARN Mensajero/química , ARN Mensajero/genéticaRESUMEN
Nuclear RNA interference (RNAi) is mediated by the canonical RNAi machinery and can lead to transcriptional silencing, transcriptional activation, or modulation of alternative splicing patterns. These effects transpire through changes in histone and DNA modifications via RNAi-mediated recruitment of chromatin-modifying enzymes. To prove that nuclear RNAi occurs and modulates transcription in human cells, we used live-cell imaging to detect and track nuclear RNAi transcriptional repression in single living human cells. While employing reporter genes constructed with inducible promoters and cognate-inducible short hairpin RNA (shRNA) targeted against the reporter coding region, we have characterized the dynamics of the nuclear RNAi process in living human cells. We show that the silencing effect is mediated through the nascent mRNA, followed by activity of histone methylating enzymes, but not through DNA methylation.
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Proteínas Fluorescentes Verdes/genética , Imagen Molecular/métodos , Interferencia de ARN , Núcleo Celular/genética , Metilación de ADN/efectos de los fármacos , Epigénesis Genética , Regulación de la Expresión Génica , Proteínas Fluorescentes Verdes/análisis , Proteínas Fluorescentes Verdes/metabolismo , Células HEK293 , Histona Metiltransferasas , N-Metiltransferasa de Histona-Lisina/antagonistas & inhibidores , N-Metiltransferasa de Histona-Lisina/genética , N-Metiltransferasa de Histona-Lisina/metabolismo , Humanos , Hibridación Fluorescente in Situ/métodos , Microscopía Fluorescente/métodos , Piperazinas/farmacología , Regiones Promotoras Genéticas , Quinazolinas/farmacología , Precursores del ARN/genética , ARN Interferente Pequeño , Sitio de Iniciación de la TranscripciónRESUMEN
Transcribed mRNA molecules must reach the cytoplasm to undergo translation. Technological developments in imaging have placed mRNAs under the spotlight, allowing the quantitative study of the spatial and temporal dynamics of the nucleocytoplasmic mRNA export process. Here, we discuss studies that have used such experimental approaches to demonstrate that gene tethering at the nuclear pore complex (NPC) regulates mRNA expression, and to characterize mRNA dynamics during transport in real time. The paths taken by mRNAs as they move from their sites of transcription and travel through the nucleoplasm, in between chromatin domains, and finally through the NPC, can now be observed in detail.
Asunto(s)
Transporte Activo de Núcleo Celular/genética , Poro Nuclear/genética , ARN Mensajero/genética , Transcripción Genética , Núcleo Celular/genética , Cromatina/genética , Citoplasma/genética , Regulación de la Expresión Génica , Transporte de ARN/genética , Ribonucleoproteínas/genéticaRESUMEN
Nuclear export of messenger RNA (mRNA) occurs by translocation of mRNA/protein complexes (mRNPs) through nuclear pore complexes (NPCs). The DEAD-box protein Dbp5 mediates export by triggering removal of mRNP proteins in a spatially controlled manner. This requires Dbp5 interaction with Nup159 in NPC cytoplasmic filaments and activation of Dbp5's ATPase activity by Gle1 bound to inositol hexakisphosphate (IP(6)). However, the precise sequence of events within this mechanism has not been fully defined. Here we analyze dbp5 mutants that alter ATP binding, ATP hydrolysis, or RNA binding. We found that ATP binding and hydrolysis are required for efficient Dbp5 association with NPCs. Interestingly, mutants defective for RNA binding are dominant-negative (DN) for mRNA export in yeast and human cells. We show that the DN phenotype stems from competition with wild-type Dbp5 for Gle1 at NPCs. The Dbp5-Gle1 interaction is limiting for export and, importantly, can be independent of Nup159. Fluorescence recovery after photobleaching experiments in yeast show a very dynamic association between Dbp5 and NPCs, averaging <1 sec, similar to reported NPC translocation rates for mRNPs. This work reveals critical steps in the Gle1-IP(6)/Dbp5/Nup159 cycle, and suggests that the number of remodeling events mediated by a single Dbp5 is limited.
Asunto(s)
Núcleo Celular/metabolismo , ARN Helicasas DEAD-box/genética , ARN Helicasas DEAD-box/metabolismo , Poro Nuclear/metabolismo , Proteínas de Transporte Nucleocitoplasmático/genética , Proteínas de Transporte Nucleocitoplasmático/metabolismo , ARN Mensajero/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Transporte Activo de Núcleo Celular , Adenosina Trifosfato/metabolismo , Línea Celular Tumoral , Células HeLa , Humanos , Hidrólisis , Mutación , Proteínas de Complejo Poro Nuclear/metabolismo , Fenotipo , Unión Proteica/genética , Saccharomyces cerevisiae/crecimiento & desarrolloRESUMEN
The transcriptional kinetics of RNA polymerase II, the enzyme responsible for mRNA transcription in the nucleoplasm, can be modulated by a variety of factors. It is therefore important to establish experimental systems that will enable the readout of transcription kinetics of specific genes as they occur in real time within individual cells. This can be performed by implementing fluorescent tagging of the mRNA under live-cell conditions. This chapter describes how to generate fluorescently tagged genes and mRNA, and how a photobleaching approach can produce information on mRNA transcription kinetics.
Asunto(s)
Recuperación de Fluorescencia tras Fotoblanqueo/métodos , Imagen Molecular/métodos , ARN Polimerasa II/metabolismo , ARN Mensajero/química , Transcripción Genética , Técnicas de Cultivo de Célula/instrumentación , Técnicas de Cultivo de Célula/métodos , Línea Celular , Núcleo Celular/metabolismo , Recuperación de Fluorescencia tras Fotoblanqueo/instrumentación , Genes Reporteros , Proteínas Fluorescentes Verdes/química , Proteínas Fluorescentes Verdes/genética , Humanos , Cinética , Microscopía Confocal/instrumentación , Microscopía Confocal/métodos , Imagen Molecular/instrumentación , Fotoblanqueo , Plásmidos/genética , ARN Polimerasa II/química , ARN Mensajero/genética , Imagen de Lapso de Tiempo/instrumentación , Imagen de Lapso de Tiempo/métodosRESUMEN
The cyanobacterial light-harvesting complex, the phycobilisome, is degraded under nutrient limitation, allowing the cell to adjust light absorbance to its metabolic capacity. This large light-harvesting antenna comprises a core complex of the pigment allophycocyanin, and rod-shaped pigment assemblies emanating from the core. NblA, a low-molecular-weight protein, is essential for degradation of the phycobilisome. NblA mutants exhibit high absorbance of rod pigments under conditions that generally elicit phycobilisome degradation, implicating NblA in degradation of these pigments. However, the vast abundance of rod pigments and the substantial overlap between the absorbance spectra of rod and core pigments has made it difficult to directly associate NblA with proteolysis of the phycobilisome core. Furthermore, lack of allophycocyanin degradation in an NblA mutant may reflect a requirement for rod degradation preceding core degradation, and does not prove direct involvement of NblA in proteolysis of the core pigment. Therefore, in this study, we used a mutant lacking phycocyanin, the rod pigment of Synechococcus elongatusPCC7942, to examine whether NblA is required for allophycocyanin degradation. We demonstrate that NblA is essential for degradation of the core complex of the phycobilisome. Furthermore, fluorescence lifetime imaging microscopy provided in situ evidence for the interaction of NblA with allophycocyanin, and indicated that NblA interacts with allophycocyanin complexes that are associated with the photosynthetic membranes. Based on these data, as well as previous observations indicating interaction of NblA with phycobilisomes attached to the photosynthetic membranes, we suggest a model for sequential phycobilisome disassembly by NblA.