RESUMO
Continued progress has been made during the past year in understanding the basic biochemical mechanisms involved in nuclear RNA processing. Of particular importance have been the advances made in purifying and characterizing protein factors involved in splicing and polyadenylation of pre-mRNAs.
Assuntos
Núcleo Celular/fisiologia , Precursores de RNA/genética , Splicing de RNA , RNA/genética , Transcrição Gênica , Animais , Sequência de Bases , Humanos , Modelos Genéticos , RNA/metabolismo , Ribonucleoproteínas/genética , Ribonucleoproteínas/metabolismo , Ribonucleoproteínas Nucleares PequenasRESUMO
The splicing of mRNA precursors (pre-mRNA) in the nucleus is catalyzed by a complex machinery termed the spliceosome. In order to understand how it functions in vivo, it is essential to complement biochemical analyses with a detailed study of how spliceosome components are organized within the nucleus.
Assuntos
Núcleo Celular/metabolismo , Precursores de RNA/metabolismo , Splicing de RNA , Spliceossomos/metabolismo , Animais , Núcleo Celular/genética , Humanos , Precursores de RNA/genética , RNA Nuclear Pequeno/metabolismoRESUMO
New developments in biological mass spectrometry and the growth of sequence databases are revolutionizing protein analysis. As recent results demonstrate, the high sensitivity and high throughput of this approach allow the rapid characterization of gel-separated proteins and the identification of cognate cDNA and expressed-sequence tag (EST) clones. We advocate here exploiting this new technology for the systematic characterization of multiprotein complexes. The analysis of proteins as components of specific complexes provides an immediate link to biological function and is a powerful method for deciphering the functions of open reading frames uncovered in the genome-sequencing projects.
RESUMO
The coiled body is a nuclear organelle that contains snRNPs involved in splicing, the non-snRNP splicing factor U2AF and the nucleolar protein fibrillarin. It is highly conserved in evolution and is present in both animal and plant cells. The coiled body is a dynamic structure that can undergo regulated cycles of assembly and disassembly during interphase and mitosis and it may represent a distinct metabolic compartment within the nucleus.
RESUMO
Coiled bodies (CBs) are nuclear organelles in which splicing snRNPs concentrate. While CBs are sometimes observed in association with the nucleolar periphery, they are shown not to contain 5S or 28S rRNA or the U3 snoRNA. This argues against CBs playing a role in rRNA maturation or transport as previously suggested. We present evidence here that CBs are kinetic structures and demonstrate that the formation of snRNP-containing CBs is regulated in interphase and mitosis. The coiled body antigen, p80 coilin, was present in all cell types studied, even when CBs were not prominent. Striking changes in the formation of CBs could be induced by changes in cellular growth temperature without a concomitant change in the intracellular p80 coilin level. During mitosis, CBs disassemble, coinciding with a mitotic-specific phosphorylation of p80 coilin. Coilin is shown to be a phosphoprotein that is phosphorylated on at least two additional sites during mitosis. CBs reform in daughter nuclei after a lag period during which they are not detected. CBs are thus, dynamic nuclear organelles and we propose that cycling interactions of splicing snRNPs with CBs may be important for their participation in the processing or transport of pre-mRNA in mammalian cells.
Assuntos
Núcleo Celular/ultraestrutura , Interfase , Mitose , Proteínas Nucleares/metabolismo , Fosfoproteínas/metabolismo , Ribonucleoproteínas Nucleares Pequenas/metabolismo , Animais , Sequência de Bases , Nucléolo Celular/metabolismo , Nucléolo Celular/ultraestrutura , Proteínas Cromossômicas não Histona/metabolismo , Imunofluorescência , Células HeLa , Humanos , Dados de Sequência Molecular , Oligonucleotídeos/química , Fosforilação , RNA/metabolismo , Splicing de RNA , RNA Nuclear Pequeno/metabolismo , Ratos , TemperaturaRESUMO
Coiled bodies are conserved subnuclear domains found in both plant and animal cells. They contain a subset of splicing snRNPs and several nucleolar antigens, including Nopp140 and fibrillarin. In addition, autoimmune patient sera have identified a coiled body specific protein, called p80 coilin. In this study we show that p80 coilin is ubiquitously expressed in human tissues. The full-length human p80 coilin protein correctly localizes in coiled bodies when exogenously expressed in HeLa cells using a transient transfection assay. Mutational analysis identifies separate domains in the p80 coilin protein that differentially affect its subnuclear localization. The data show that p80 coilin has a nuclear localization signal, but this is not sufficient to target the protein to coiled bodies. The results indicate that localization in coiled bodies is not determined by a simple motif analogous to the NLS motifs involved in nuclear import. A specific carboxy-terminal deletion in p80 coilin results in the formation of pseudo-coiled bodies that are unable to recruit splicing snRNPs. This causes a loss of endogenous coiled bodies. A separate class of mutant coilin proteins are shown to localize in fibrillar structures that surround nucleoli. These mutants also lead to loss of endogenous coiled bodies, produce a dramatic disruption of nucleolar architecture and cause a specific segregation of nucleolar antigens. The structural change in nucleoli is accompanied by the loss of RNA polymerase I activity. These data indicate that p80 coilin plays an important role in subnuclear organization and suggest that there may be a functional interaction between coiled bodies and nucleoli.
Assuntos
Autoantígenos/genética , Nucléolo Celular/fisiologia , Proteínas Nucleares/genética , Organelas/fisiologia , Sequência de Aminoácidos , Nucléolo Celular/imunologia , Nucléolo Celular/ultraestrutura , Proteínas Cromossômicas não Histona/genética , Proteínas Cromossômicas não Histona/metabolismo , Análise Mutacional de DNA , Deleção de Genes , Células HeLa , Humanos , Microscopia Imunoeletrônica , Dados de Sequência Molecular , Organelas/ultraestrutura , Ribonucleoproteínas Nucleares Pequenas/genética , Ribonucleoproteínas Nucleares Pequenas/metabolismo , TransfecçãoRESUMO
Murine erythroleukemia (MEL) cells are erythroid progenitors that can be induced to undergo terminal erythroid differentiation in culture. We have used MEL cells here as a model system to study the nuclear organization of splicing snRNPs during the physiological changes in gene expression which accompany differentiation. In uninduced MEL cells, snRNPs are widely distributed throughout the nucleoplasm and show an elevated concentration in coiled bodies. Within the first two days after induction of terminal erythroid differentiation, the pattern of gene expression changes, erythroid-specific transcription is activated and transcription of many other genes is repressed. During this early stage splicing snRNPs remain widely distributed through the nucleoplasm and continue to associate with coiled bodies. At later stages of differentiation (four to six days), when total transcription levels have greatly decreased, splicing snRNPs are redistributed. By six days postinduction snRNPs were concentrated in large clusters of interchromatin granules and no longer associated with coiled bodies. At the end-point of erythroid differentiation, just before enucleation, we observe a dramatic segregation of splicing snRNPs from the condensed chromatin. Analysis by EM shows that the snRNPs are packaged into a membrane-associated structure at the nuclear periphery which we term the "SCIM" domain (i.e., SnRNP Clusters Inside a Membrane).
Assuntos
Núcleo Celular/ultraestrutura , Células Precursoras Eritroides/ultraestrutura , Eritropoese/fisiologia , Splicing de RNA , Ribonucleoproteínas Nucleares Pequenas/metabolismo , Ribonucleoproteínas , Animais , Compartimento Celular , Núcleo Celular/fisiologia , Cromatina/ultraestrutura , Células Precursoras Eritroides/fisiologia , Imunofluorescência , Expressão Gênica , Hibridização In Situ , Corpos de Inclusão , Camundongos , Microscopia Eletrônica , Proteínas Nucleares/isolamento & purificação , Ribonucleoproteínas Nucleares Pequenas/ultraestrutura , Fatores de Processamento de Serina-Arginina , Células Tumorais CultivadasRESUMO
In the interphase nucleus of mammalian cells the U1, U2, U4/U6, and U5 small nuclear ribonucleoproteins (snRNPs), which are subunits of spliceosomes, associate with specific subnuclear domains including interchromatin granules and coiled bodies. Here, we analyze the association of splicing snRNPs with these structures during mitosis and reassembly of daughter nuclei. At the onset of mitosis snRNPs are predominantly diffuse in the cytoplasm, although a subset remain associated with remnants of coiled bodies and clusters of mitotic interchromatin granules, respectively. The number and size of mitotic coiled bodies remain approximately unchanged from metaphase to early telophase while snRNP-containing clusters of mitotic interchromatin granules increase in size and number as cells progress from anaphase to telophase. During telophase snRNPs are transported into daughter nuclei while the clusters of mitotic interchromatin granules remain in the cytoplasm. The timing of nuclear import of splicing snRNPs closely correlates with the onset of transcriptional activity in daughter nuclei. When transcription restarts in telophase cells snRNPs have a diffuse nucleoplasmic distribution. As cells progress to G1 snRNP-containing clusters of interchromatin granules reappear in the nucleus. Coiled bodies appear later in G1, although the coiled body antigen, p80 coilin, enters early into telophase nuclei. After inhibition of transcription we still observe nuclear import of snRNPs and the subsequent appearance of snRNP-containing clusters of interchromatin granules, but not coiled body formation. These data demonstrate that snRNP associations with coiled bodies and interchromatin granules are differentially regulated during the cell division cycle and suggest that these structures play distinct roles connected with snRNP structure, transport, and/or function.
Assuntos
Compartimento Celular , Núcleo Celular/fisiologia , Cromatina/fisiologia , Mitose/fisiologia , Splicing de RNA , Ribonucleoproteínas Nucleares Pequenas/metabolismo , Transporte Biológico/efeitos dos fármacos , Núcleo Celular/ultraestrutura , Células Cultivadas , Cromatina/ultraestrutura , Citoplasma/metabolismo , Citoplasma/ultraestrutura , Dactinomicina/farmacologia , Imunofluorescência , Humanos , Hibridização In Situ , Ribonucleoproteína Nuclear Pequena U1/isolamento & purificação , Ribonucleoproteína Nuclear Pequena U1/metabolismo , Ribonucleoproteína Nuclear Pequena U2/isolamento & purificação , Ribonucleoproteína Nuclear Pequena U2/metabolismo , Ribonucleoproteínas Nucleares Pequenas/isolamento & purificação , Spliceossomos/metabolismo , Telófase/fisiologia , Transcrição GênicaRESUMO
Cajal bodies (also known as coiled bodies) are subnuclear organelles that contain specific nuclear antigens, including splicing small nuclear ribonucleoproteins (snRNPs) and a subset of nucleolar proteins. Cajal bodies are localized in the nucleoplasm and are often found at the nucleolar periphery. We have constructed a stable HeLa cell line, HeLa(GFP-coilin), that expresses the Cajal body marker protein, p80 coilin, fused to the green fluorescent protein (GFP-coilin). The localization pattern and biochemical properties of the GFP-coilin fusion protein are identical to the endogenous p80 coilin. Time-lapse recordings on 63 nuclei of HeLa(GFP-coilin) cells showed that all Cajal bodies move within the nucleoplasm. Movements included translocations through the nucleoplasm, joining of bodies to form larger structures, and separation of smaller bodies from larger Cajal bodies. Also, we observed Cajal bodies moving to and from nucleoli. The data suggest that there may be at least two classes of Cajal bodies that differ in their size, antigen composition, and dynamic behavior. The smaller size class shows more frequent and faster rates of movement, up to 0.9 microm/min. The GFP-coilin protein is dynamically associated with Cajal bodies as shown by changes in their fluorescence intensity over time. This study reveals an unexpectedly high level of movement and interactions of nuclear bodies in human cells and suggests that these movements may be driven, at least in part, by regulated mechanisms.
Assuntos
Corpos Enovelados/metabolismo , Movimento , Transporte Biológico , Western Blotting , Nucléolo Celular/metabolismo , Sobrevivência Celular , Corpos Enovelados/classificação , Imunofluorescência , Células HeLa , Humanos , Microscopia de Fluorescência , Microscopia de Vídeo , Matriz Nuclear/metabolismo , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Ligação Proteica , Proteínas Recombinantes de Fusão/metabolismo , Fatores de TempoRESUMO
We have analyzed the spatial organization of large scale chromatin domains in chinese hamster fibroblast, human lymphoid (IM-9), and marsupial kidney epithelial (PtK) cells by labeling DNA at defined stages of S phase via pulsed incorporation of halogenated deoxynucleosides. Most, if not all, chromosomes contribute multiple chromatin domains to both peripheral and internal nucleoplasmic compartments. The peripheral compartment contains predominantly late replicating G/Q bands, whereas early replicating R bands preferentially localize to the internal nucleoplasmic compartment. During mitosis, the labeled chromatin domains that were separated in interphase form a pattern of intercalated bands along the length of each metaphase chromosome. The transition from a banded (mitotic) to a compartmentalized (interphasic) organization of chromatin domains occurs during the late telophase/early G1 stage and is independent of transcriptional activation of the genome. Interestingly, generation of micronuclei with a few chromosomes showed that the spatial separation of early and late replicating chromatin compartments is recapitulated independently of chromosome number, even in micronuclei containing only a single chromosome. Our data strongly support the notion that the compartmentalization of large-scale (band size) chromatin domains seen in the intact nucleus is a magnified image of a similar compartmentalization occurring in individual chromosome territories.
Assuntos
Cromatina , Cromossomos Humanos/ultraestrutura , Cromossomos/ultraestrutura , DNA/análise , Animais , Compartimento Celular , Linhagem Celular , Núcleo Celular/metabolismo , Cromatina/ultraestrutura , Bandeamento Cromossômico , Cromossomos/química , Cromossomos Humanos/química , Cricetinae , Replicação do DNA , Humanos , Interfase , MarsupiaisRESUMO
We have recently shown that discrete foci are present in the nuclei of mammalian cells in which each of the U1, U2, U4/U6, and U5 snRNPs involved in pre-mRNA splicing, and the non-snRNP-splicing factor U2AF, are concentrated (Carmo-Fonseca, M., D. Tollervey, R. Pepperkok, S. Barabino, A. Merdes, C. Brunner, P. D. Zamore, M. R. Green, E. Hurt, and A. I. Lamond. 1991. EMBO (Eur. Mol. Biol. Organ.) J. 10:195-206; Carmo-Fonseca, M., R. Pepperkok, B. S. Sproat, W. Ansorge, M. S. Swanson, and A. I. Lamond. 1991 EMBO (Eur. Mol. Biol. Organ.) J. 10:1863-1873). Here, we identify these snRNP-rich organelles as coiled bodies. snRNPs no longer concentrate in coiled bodies after cells are treated with the transcription inhibitors alpha-amanitin or actinomycin D. snRNP association with coiled bodies is also disrupted by heat shock. This indicates that the association of snRNPs with coiled bodies may be connected with the metabolism of nascent transcripts. A novel labeling method is described which shows both the RNA and protein components of individual snRNPs colocalizing in situ. Using this procedure all spliceosomal snRNPs are seen distributed in a nonhomogeneous pattern throughout the nucleoplasm, excluding nucleoli. They are most concentrated in coiled bodies, but in addition are present in "speckled" structures which are distinct from coiled bodies and which contain the non-snRNP splicing factor SC-35. U1 snRNP shows a more widespread nucleoplasmic staining, outside of coiled bodies and "speckled" structures, relative to the other snRNPs. The association of snRNPs with "speckles" is disrupted by heat shock but enhanced when cells are treated with alpha-amanitin.
Assuntos
Organelas/ultraestrutura , RNA Nuclear Pequeno/genética , Ribonucleoproteínas/genética , Transcrição Gênica , Amanitinas/farmacologia , Elementos Antissenso (Genética) , Sequência de Bases , Núcleo Celular/fisiologia , Núcleo Celular/ultraestrutura , Dactinomicina/farmacologia , Células HeLa , Humanos , Dados de Sequência Molecular , Oligodesoxirribonucleotídeos , Organelas/efeitos dos fármacos , Organelas/fisiologia , Ribonucleoproteínas/análise , Ribonucleoproteínas Nucleares Pequenas , Transcrição Gênica/efeitos dos fármacosRESUMO
Import of proteins into the nucleus is a two-step process, involving nuclear localization sequence (NLS)-dependent docking of the substrate at the nuclear envelope followed by translocation through the nuclear pore. A recombinant human protein, hSRP1 alpha, bound in vitro specifically and directly to substrates containing either a simple or bipartite NLS motif. hSRP1 alpha promoted docking of import substrates to the nuclear envelope and together with recombinant human Ran reconstituted complete nuclear protein import. Thus, hSRP1 alpha has the properties of a cytosolic receptor for both simple and bipartite NLS motifs.
Assuntos
Núcleo Celular/metabolismo , Proteínas Nucleares/fisiologia , Fosfoproteínas/fisiologia , Sequência de Aminoácidos , Sequência de Bases , Ligação Competitiva , Transporte Biológico/fisiologia , Células HeLa , Humanos , Dados de Sequência Molecular , Família Multigênica , Proteínas Nucleares/genética , Fosfoproteínas/genética , Ligação Proteica , Sinais Direcionadores de Proteínas/metabolismo , Proteínas Recombinantes , Homologia de Sequência de AminoácidosRESUMO
Current evidence suggests that the nucleus has a distinct substructure, albeit one that is dynamic rather than a rigid framework. Viral infection, oncogene expression, and inherited human disorders can each cause profound and specific changes in nuclear organization. This review summarizes recent progress in understanding nuclear organization, highlighting in particular the dynamic aspects of nuclear structure.
Assuntos
Núcleo Celular/fisiologia , Núcleo Celular/ultraestrutura , Proteínas de Drosophila , Proteínas Nucleares , Animais , Nucléolo Celular/fisiologia , Nucléolo Celular/ultraestrutura , Núcleo Celular/química , Cromatina/fisiologia , Cromossomos/fisiologia , Eucromatina , Regulação da Expressão Gênica , Heterocromatina/fisiologia , Humanos , Proteínas de Insetos/química , Proteínas de Insetos/fisiologia , Interfase , Proteínas de Neoplasias/química , Proteínas de Neoplasias/fisiologia , Complexo Repressor Polycomb 1 , Proteínas do Grupo Polycomb , Proteína da Leucemia Promielocítica , Proteínas Repressoras/química , Proteínas Repressoras/fisiologia , Ribonucleoproteínas Nucleares Pequenas/análise , Ribonucleoproteínas Nucleares Pequenas/fisiologia , Fatores de Transcrição/química , Fatores de Transcrição/fisiologia , Proteínas Supressoras de TumorRESUMO
Major isoforms of WT1--products of the tumour suppressor gene WT1, implicated in predisposition to Wilms' tumour--may preferentially interact with splicing factors, suggesting a role for WT1 in RNA processing.
Assuntos
Proteínas de Ligação a DNA/genética , Genes do Tumor de Wilms , Neoplasias Renais/genética , Splicing de RNA , Fatores de Transcrição/genética , Tumor de Wilms/genética , Animais , Sequência de Bases , Humanos , Dados de Sequência Molecular , Processamento Pós-Transcricional do RNA , Proteínas WT1RESUMO
A novel form of spliceosome, containing the minor snRNPs U11 and U12, splices a class of pre-mRNA introns with non-consensus splice sites. This unexpected spliceosome diversity has interesting implications for the evolution and expression of eukaryotic genes.
Assuntos
Íntrons , Proteínas Nucleares/genética , Precursores de RNA , Splicing de RNA , Ribonucleoproteínas Nucleares Pequenas/metabolismo , Animais , Humanos , Spliceossomos , tRNA MetiltransferasesRESUMO
Splicing factors belonging to the DEAD-box superfamily can affect RNA conformation in vitro. These proteins may control the dynamic changes in RNA base-pairing interactions during spliceosome assembly.
Assuntos
Splicing de RNA , Spliceossomos/fisiologia , Sequência de Aminoácidos , Animais , Humanos , Dados de Sequência Molecular , RNA Nucleotidiltransferases/fisiologiaRESUMO
BACKGROUND: Small nuclear ribonucleoproteins (snRNPs), which are essential components of the mRNA splicing machinery, comprise small nuclear RNAs, each complexed with a set of proteins. An early event in the maturation of snRNPs is the binding of the core proteins - the Sm proteins - to snRNAs in the cytoplasm followed by nuclear import. Immunolabelling with antibodies against Sm proteins shows that splicing snRNPs have a complex steady-state localisation within the nucleus, the result of the association of snRNPs with several distinct subnuclear structures. These include speckles, coiled bodies and nucleoli, in addition to a diffuse nucleoplasmic compartment. The reasons for snRNP accumulation in these different structures are unclear. RESULTS: When mammalian cells were microinjected with plasmids encoding the Sm proteins B, D1 and E, each tagged with either the green fluorescent protein (GFP) or yellow-shifted GFP (YFP), a pulse of expression of the tagged proteins was observed. In each case, the newly synthesised GFP/YFP-labelled snRNPs accumulated first in coiled bodies and nucleoli, and later in nuclear speckles. Mature snRNPs localised immediately to speckles upon entering the nucleus after cell division. CONCLUSIONS: The complex nuclear localisation of splicing snRNPs results, at least in part, from a specific pathway for newly assembled snRNPs. The data demonstrate that the distribution of snRNPs between coiled bodies and speckles is directed and not random.
Assuntos
Núcleo Celular/metabolismo , Corpos Enovelados/metabolismo , Ribonucleoproteínas Nucleares Pequenas/metabolismo , Proteínas de Saccharomyces cerevisiae , Autoantígenos/metabolismo , Proteínas de Bactérias/metabolismo , Nucléolo Celular/fisiologia , Proteínas de Fluorescência Verde , Células HeLa , Humanos , Cinética , Proteínas Luminescentes/metabolismo , Microscopia Confocal , Proteínas Recombinantes de Fusão/metabolismo , Fatores de Tempo , Células Tumorais Cultivadas , Proteínas Centrais de snRNPRESUMO
The recent discovery of catalytic RNA molecules has provoked enormous interest in the origin of life and has given rise to new speculations about how living systems developed on the primitive earth. Here we outline why the discovery of catalytic RNA molecules has profound evolutionary implications, and then go on to discuss models for the emergence of metabolic complexity and protein synthesis in a primitive 'RNA world', emphasizing the constraints imposed on such models by genetical arguments.
Assuntos
Evolução Biológica , Precursores de RNA/metabolismo , RNA Ribossômico/metabolismo , Histidina/biossíntese , Estrutura Molecular , Biossíntese de Proteínas , Proteínas/metabolismo , Precursores de RNA/genética , RNA Catalítico , RNA Ribossômico/genéticaRESUMO
The coiled body is a specific intranuclear structure of unknown function that is enriched in splicing small nuclear ribonucleoproteins (snRNPs). Because adenoviruses make use of the host cell-splicing machinery and subvert the normal subnuclear organization, we initially decided to investigate the effect of adenovirus infection on the coiled body. The results indicate that adenovirus infection induces the disassembly of coiled bodies and that this effect is probably secondary to the block of host protein synthesis induced by the virus. Furthermore, coiled bodies are shown to be very labile structures, with a half-life of approximately 2 h after treatment of HeLa cells with protein synthesis inhibitors. After blocking of protein synthesis, p80 coilin was detected in numerous microfoci that do not concentrate snRNP. These structures may represent precursor forms of the coiled body, which goes through a rapid cycle of assembly/disassembly in the nucleus and requires ongoing protein synthesis to reassemble.
Assuntos
Infecções por Adenovirus Humanos/patologia , Adenovírus Humanos/patogenicidade , Núcleo Celular/ultraestrutura , Infecções por Adenovirus Humanos/metabolismo , Adenovírus Humanos/metabolismo , Sequência de Bases , Núcleo Celular/metabolismo , Núcleo Celular/virologia , Meia-Vida , Células HeLa , Humanos , Hibridização In Situ , Microscopia Imunoeletrônica , Sondas Moleculares/genética , Proteínas Nucleares/metabolismo , Inibidores da Síntese de Proteínas/farmacologia , RNA Viral/metabolismo , Ribonucleoproteínas Nucleares Pequenas/metabolismoRESUMO
Mammalian proteins expressed in Escherichia coli are used in a variety of applications. A major drawback in producing eukaryotic proteins in E.coli is that the bacteria lack most eukaryotic post-translational modification systems, including serine/threonine protein kinase(s). Here we show that a eukaryotic protein can be phosphorylated in E.coli by simultaneous expression of a mammalian protein kinase and its substrate. We show that in bacteria expressing SRPK1, ASF/SF2 becomes phosphorylated to a degree resembling native ASF/SF2 present in interphase HeLa cell nuclei. The E.coli phosphorylated ASF/SF2 is functional in splicing and, contrary to the unphosphorylated protein, soluble under native conditions.