RESUMO
Proteins, RNAs and even large macromolecular complexes are transported into and out of nuclei with remarkable rapidity and specificity. Nucleocytoplasmic transport must therefore be efficient and selective. Characterization of the roles of the importin beta family of transport receptors and of the Ran GTPase has showed how these characteristics can be achieved, but there are many examples of nucleocytoplasmic transport that do not fit this model. Here, we discuss current understanding of various transport mechanisms and evaluate cases in which the molecules and mechanisms underlying nucleocytoplasmic transport are used to carry out important cellular functions in the absence of a nucleus.
Assuntos
Núcleo Celular/metabolismo , Citoplasma/metabolismo , Proteínas Nucleares/metabolismo , Proteínas de Transporte Nucleocitoplasmático , Proteínas de Ligação a RNA/metabolismo , beta Carioferinas/metabolismo , Proteína ran de Ligação ao GTP/metabolismo , Transporte Ativo do Núcleo Celular/fisiologia , Animais , Cromossomos/metabolismo , Humanos , Proteínas Ribossômicas/metabolismo , Ribossomos/metabolismo , Fatores de Transcrição/metabolismoRESUMO
The nuclear pore complex (NPC) is a large proteinaceous structure through which bidirectional transport of macromolecules across the nuclear envelope (NE) takes place. Nup153 is a peripheral NPC component that has been implicated in protein and RNP transport and in the interaction of NPCs with the nuclear lamina. Here, Nup153 is localized by immunogold electron microscopy to a position on the nuclear ring of the NPC. Nuclear reconstitution is used to investigate the role of Nup153 in nucleo- cytoplasmic transport and NPC architecture. NPCs assembled in the absence of Nup153 lacked several nuclear basket components, were unevenly distributed in the NE and, unlike wild-type NPCs, were mobile within the NE. Importin alpha/beta-mediated protein import into the nucleus was strongly reduced in the absence of Nup153, while transportin-mediated import was unaffected. This was due to a reduction in import complex translocation rather than to defective receptor recycling. Our results therefore reveal functions for Nup153 in NPC assembly, in anchoring NPCs within the NE and in mediating specific nuclear import events.
Assuntos
Complexo de Proteínas Formadoras de Poros Nucleares/fisiologia , Poro Nuclear/fisiologia , Proteínas Nucleares/metabolismo , Transporte Proteico/fisiologia , Animais , Bovinos , Feminino , Ribonucleoproteínas Nucleares Heterogêneas , Humanos , Imuno-Histoquímica , Carioferinas/metabolismo , Substâncias Macromoleculares , Masculino , Microscopia Imunoeletrônica , Poro Nuclear/ultraestrutura , Proteínas Nucleares/genética , Nucleoplasminas , Oócitos , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Ribonucleoproteínas/genética , Ribonucleoproteínas/metabolismo , Soroalbumina Bovina/genética , Soroalbumina Bovina/metabolismo , Xenopus laevisRESUMO
The role of herpes simplex virus ICP27 protein in mRNA export is investigated by microinjection into Xenopus laevis oocytes. ICP27 dramatically stimulates the export of intronless viral mRNAs, but has no effect on the export of cellular mRNAs, U snRNAs or tRNA. Use of inhibitors shows, in contrast to previous suggestions, that ICP27 neither shuttles nor exports viral mRNA via the CRM1 pathway. Instead, ICP27-mediated viral RNA export requires REF and TAP/NXF1, factors involved in cellular mRNA export. ICP27 binds directly to REF and complexes containing ICP27, REF and TAP are found in vitro and in virally infected cells. A mutant ICP27 that does not interact with REF is inactive in viral mRNA export. We propose that ICP27 associates with viral mRNAs and recruits TAP/NXF1 via its interaction with REF proteins, allowing the otherwise inefficiently exported viral mRNAs to access the TAP-mediated export pathway. This represents a novel mechanism for export of viral mRNAs.
Assuntos
Proteínas Imediatamente Precoces/fisiologia , Proteínas de Transporte Nucleocitoplasmático , RNA Mensageiro/metabolismo , RNA Viral/metabolismo , Proteínas de Saccharomyces cerevisiae , Simplexvirus/fisiologia , Animais , Transporte Biológico , Feminino , Proteínas Fúngicas/metabolismo , Regulação Viral da Expressão Gênica , Células HeLa , Humanos , Substâncias Macromoleculares , Microinjeções , Proteínas Nucleares/metabolismo , Oócitos , Ligação Proteica , Proteínas de Ligação a RNA/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Simplexvirus/genética , Xenopus laevisRESUMO
The heterodimeric nuclear cap binding complex (CBC) binds to 5'-capped polymerase II transcripts. It enhances the efficiency of several mRNA maturation steps and is essential for U snRNA nuclear export in multicellular eukaryotes. The 2A crystal structure of human CBC shows that the large subunit, CBP80, comprises three domains, each containing consecutive helical hairpins and resembling the so-called MIF4G domain found in several other proteins involved in RNA metabolism. The small subunit, CPB20, has an RNP fold and associates with the second and third domains of CBP80. Site-directed mutagenesis revealed 4 residues of CBP20 which are critical for cap binding. A model for cap binding is proposed based on these results and the known mode of binding of RNA to RNP domains.
Assuntos
Estrutura Quaternária de Proteína , Proteínas de Ligação a RNA/química , Sequência de Aminoácidos , Animais , Sítios de Ligação , Reagentes de Ligações Cruzadas/química , Reagentes de Ligações Cruzadas/metabolismo , Cristalografia por Raios X , Humanos , Modelos Moleculares , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Ligação Proteica , Conformação Proteica , Subunidades Proteicas , Proteínas de Ligação ao Cap de RNA , Capuzes de RNA/química , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Alinhamento de SequênciaRESUMO
We investigated the role of RanBP3, a nuclear member of the Ran-binding protein 1 family, in CRM1-mediated protein export in higher eukaryotes. RanBP3 interacts directly with CRM1 and also forms a trimeric complex with CRM1 and RanGTP. However, RanBP3 does not bind to CRM1 like an export substrate. Instead, it can stabilize CRM1-export substrate interaction. Nuclear RanBP3 stimulates CRM1-dependent protein export in permeabilized cells. These data indicate that RanBP3 functions by a novel mechanism as a cofactor in recognition and export of certain CRM1 substrates. In vitro, RanBP3 binding to CRM1 affects the relative affinity of CRM1 for different substrates.
Assuntos
Proteínas de Transporte/metabolismo , Proteínas de Transporte/fisiologia , Núcleo Celular/metabolismo , Carioferinas/metabolismo , Proteínas Nucleares/metabolismo , Proteínas Nucleares/fisiologia , Proteínas de Transporte Nucleocitoplasmático , Receptores Citoplasmáticos e Nucleares , Relação Dose-Resposta a Droga , GTP Fosfo-Hidrolases/metabolismo , Glutationa Transferase/metabolismo , Células HeLa , Humanos , Carioferinas/química , Cinética , Plasmídeos/metabolismo , Ligação Proteica , Especificidade por Substrato , Fatores de Tempo , Proteína ran de Ligação ao GTP/metabolismo , Proteína Exportina 1RESUMO
In metazoa, a subset of spliceosomal U snRNAs are exported from the nucleus after transcription. This export occurs in a large complex containing a U snRNA, the nuclear cap binding complex (CBC), the leucine-rich nuclear export signal receptor CRM1/Xpo1, RanGTP, and the recently identified phosphoprotein PHAX (phosphorylated adaptor for RNA export). Previous results indicated that PHAX made direct contact with RNA, CBC, and Xpo1 in the U snRNA export complex. We have now performed a systematic characterization of the functional domains of PHAX. The most evolutionarily conserved region of PHAX is shown to be a novel RNA-binding domain that is essential for U snRNA export. In addition, PHAX contains two major nuclear localization signals (NLSs) that are required for its recycling to the nucleus after export. The interaction domain of PHAX with CBC is at least partly distinct from the RNA-binding domain and the NLSs. Thus, the different interaction domains of PHAX allow it to act as a scaffold for the assembly of U snRNA export complexes.
Assuntos
Núcleo Celular/metabolismo , Sequência Conservada , Proteínas de Transporte Nucleocitoplasmático , Fosfoproteínas/genética , RNA Nuclear Pequeno/metabolismo , Proteínas de Ligação a RNA/genética , Transporte Ativo do Núcleo Celular , Sequência de Aminoácidos , Animais , Sítios de Ligação , Evolução Molecular , Feminino , Células HeLa , Humanos , Dados de Sequência Molecular , Sinais de Localização Nuclear , Oócitos , Fosfoproteínas/metabolismo , Proteínas de Ligação a RNA/metabolismo , Homologia de Sequência de Aminoácidos , XenopusRESUMO
It was recently reported that GTP-bound Ran induces microtubule and pseudo-spindle assembly in mitotic egg extracts in the absence of chromosomes and centrosomes, and that chromosomes induce the assembly of spindle microtubules in these extracts through generation of Ran-GTP. Here we examine the effects of Ran-GTP on microtubule nucleation and dynamics and show that Ran-GTP has independent effects on both the nucleation activity of centrosomes and the stability of centrosomal microtubules. We also show that inhibition of Ran-GTP production, even in the presence of duplicated centrosomes and kinetochores, prevents assembly of a bipolar spindle in M-phase extracts.
Assuntos
Microtúbulos/metabolismo , Fuso Acromático/fisiologia , Proteína ran de Ligação ao GTP/metabolismo , Animais , Centrossomo/metabolismo , Cromossomos/metabolismo , Replicação do DNA , Humanos , Masculino , Microscopia de Fluorescência , Oócitos/química , Oócitos/efeitos dos fármacos , Oócitos/metabolismo , Proteínas Proto-Oncogênicas c-mos/farmacologia , Espermatozoides/química , Espermatozoides/citologia , Xenopus laevis , Proteína ran de Ligação ao GTP/genéticaRESUMO
The small GTPase Ran, bound to GTP, is required for the induction of spindle formation by chromosomes in M phase. High concentrations of Ran.GTP are proposed to surround M phase chromatin. We show that the action of Ran.GTP in spindle formation requires TPX2, a microtubule-associated protein previously known to target a motor protein, Xklp2, to microtubules. TPX2 is normally inactivated by binding to the nuclear import factor, importin alpha, and is displaced from importin alpha by the action of Ran.GTP. TPX2 is required for Ran.GTP and chromatin-induced microtubule assembly in M phase extracts and mediates spontaneous microtubule assembly when present in excess over free importin alpha. Thus, components of the nuclear transport machinery serve to regulate spindle formation in M phase.
Assuntos
Proteínas de Ciclo Celular , Proteínas Associadas aos Microtúbulos/metabolismo , Mitose/fisiologia , Proteínas de Neoplasias , Proteínas Nucleares/metabolismo , Fosfoproteínas , Fuso Acromático/metabolismo , Proteínas de Xenopus , Proteína ran de Ligação ao GTP/metabolismo , Animais , Cromatina/metabolismo , Clonagem Molecular , Feminino , GTP Fosfo-Hidrolases/genética , GTP Fosfo-Hidrolases/isolamento & purificação , GTP Fosfo-Hidrolases/metabolismo , Expressão Gênica/fisiologia , Células HeLa , Humanos , Carioferinas , Proteínas Associadas aos Microtúbulos/genética , Proteínas Associadas aos Microtúbulos/isolamento & purificação , Microtúbulos/metabolismo , Oócitos/citologia , Oócitos/metabolismo , Xenopus laevis , Proteína ran de Ligação ao GTP/genética , Proteína ran de Ligação ao GTP/isolamento & purificaçãoRESUMO
Although nuclear envelope (NE) assembly is known to require the GTPase Ran, the membrane fusion machinery involved is uncharacterized. NE assembly involves formation of a reticular network on chromatin, fusion of this network into a closed NE and subsequent expansion. Here we show that p97, an AAA-ATPase previously implicated in fusion of Golgi and transitional endoplasmic reticulum (ER) membranes together with the adaptor p47, has two discrete functions in NE assembly. Formation of a closed NE requires the p97-Ufd1-Npl4 complex, not previously implicated in membrane fusion. Subsequent NE growth involves a p97-p47 complex. This study provides the first insights into the molecular mechanisms and specificity of fusion events involved in NE formation.
Assuntos
Adenosina Trifosfatases/metabolismo , Fusão de Membrana/fisiologia , Membrana Nuclear/enzimologia , Complexo de Proteínas Formadoras de Poros Nucleares , Proteínas Nucleares/metabolismo , Animais , Proteínas de Transporte/metabolismo , Retículo Endoplasmático/metabolismo , Complexo de Golgi/metabolismo , Proteínas de Transporte Nucleocitoplasmático , Oócitos/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Transporte Vesicular , XenopusRESUMO
The mRNA cap structure is bound by either the nuclear (CBC) or the cytoplasmic (eIF4F) cap binding complex. Following mRNA export, CBC must be exchanged for eIF4F in the cytoplasm. It is not known how this exchange occurs or how this RNP remodeling event is integrated with mRNA function. Here we report genetic and biochemical evidence that the yeast translation initiation factor eIF4G associates with CBC, and that eIF4E, the eIF4F component that binds both the cap and eIF4G, antagonizes this interaction. Furthermore, we find that CBC can stimulate translation in extracts containing an eIF4G protein deficient for eIF4E binding. These data suggest that eIF4E binding to the eIF4G-CBC complex on newly exported mRNA displaces CBC, and that the first round of translation on mRNA may occur via a different mechanism than subsequent rounds.
Assuntos
Proteínas Fúngicas/metabolismo , Fatores de Iniciação de Peptídeos/metabolismo , Capuzes de RNA/metabolismo , Proteínas de Ligação a RNA/metabolismo , Núcleo Celular/metabolismo , Fator de Iniciação Eucariótico 4G , Proteínas Fúngicas/genética , Genes Fúngicos , Mutação , Fatores de Iniciação de Peptídeos/genética , Proteínas de Ligação a Poli(A) , Biossíntese de Proteínas , Proteínas de Ligação ao Cap de RNA , RNA Fúngico/metabolismo , Proteínas de Ligação a RNA/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismoRESUMO
Nuclear formation in Xenopus egg extracts requires cytosol and is inhibited by GTP gamma S, indicating a requirement for GTPase activity. Nuclear envelope (NE) vesicle fusion is extensively inhibited by GTP gamma S and two mutant forms of the Ran GTPase, Q69L and T24N. Depletion of either Ran or RCC1, the exchange factor for Ran, from the assembly reaction also inhibits this step of NE formation. Ran depletion can be complemented by the addition of Ran loaded with either GTP or GDP but not with GTP gamma S. RCC1 depletion is only complemented by RCC1 itself or by RanGTP. Thus, generation of RanGTP by RCC1 and GTP hydrolysis by Ran are both required for the extensive membrane fusion events that lead to NE formation.
Assuntos
Proteínas de Ciclo Celular , Fatores de Troca do Nucleotídeo Guanina , Guanosina Trifosfato/metabolismo , Membrana Nuclear/metabolismo , Proteínas Nucleares , Xenopus laevis , Proteína ran de Ligação ao GTP/metabolismo , Substituição de Aminoácidos , Animais , Cromatina/química , Cromatina/metabolismo , Proteínas de Ligação a DNA/metabolismo , Feminino , Imunofluorescência , Corantes Fluorescentes , Guanosina 5'-O-(3-Tiotrifosfato)/metabolismo , Guanosina 5'-O-(3-Tiotrifosfato)/farmacologia , Guanosina Difosfato/metabolismo , Guanosina Trifosfato/farmacologia , Hidrólise/efeitos dos fármacos , Masculino , Fusão de Membrana/efeitos dos fármacos , Mutação , Membrana Nuclear/efeitos dos fármacos , Oócitos/citologia , Oócitos/metabolismo , Solubilidade , Cabeça do Espermatozoide/metabolismo , Proteínas de Xenopus , Proteína ran de Ligação ao GTP/genéticaRESUMO
In metazoa, assembly of spliceosomal U snRNPs requires nuclear export of U snRNA precursors. Export depends upon the RNA cap structure, nuclear cap-binding complex (CBC), the export receptor CRM1/Xpo1, and RanGTP. These components are however insufficient to support U snRNA export. We identify PHAX (phosphorylated adaptor for RNA export) as the additional factor required for U snRNA export complex assembly in vitro. In vivo, PHAX is required for U snRNA export but not for CRM1-mediated export in general. PHAX is phosphorylated in the nucleus and then exported with RNA to the cytoplasm, where it is dephosphorylated. PHAX phosphorylation is essential for export complex assembly while its dephosphorylation causes export complex disassembly. The compartmentalized PHAX phosphorylation cycle can contribute to the directionality of export.
Assuntos
Carioferinas , Membrana Nuclear/genética , Membrana Nuclear/metabolismo , Proteínas de Transporte Nucleocitoplasmático , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , RNA Nuclear Pequeno/genética , RNA Nuclear Pequeno/metabolismo , Receptores Citoplasmáticos e Nucleares , Sequência de Aminoácidos , Animais , Proteínas de Transporte/metabolismo , Compartimento Celular/fisiologia , Guanosina Trifosfato/metabolismo , Técnicas In Vitro , Dados de Sequência Molecular , Oócitos/fisiologia , Fosforilação , Proteínas de Ligação a RNA , Coelhos , Reticulócitos , Xenopus , Proteína ran de Ligação ao GTP/metabolismo , Proteína Exportina 1RESUMO
The status of the poly(A) tail at the 3'-end of mRNAs controls the expression of numerous genes in response to developmental and extracellular signals. Poly(A) tail regulation requires cooperative binding of two human U1A proteins to an RNA regulatory region called the polyadenylation inhibition element (PIE). When bound to PIE RNA, U1A proteins also bind to the enzyme responsible for formation of the mature 3'-end of most eukaryotic mRNAs, poly(A) polymerase (PAP). The NMR structure of the 38 kDa complex formed between two U1A molecules and PIE RNA shows that binding cooperativity depends on helix C located at the end of the RNA-binding domain and just adjacent to the PAP-interacting domain of U1A. Since helix C undergoes a conformational change upon RNA binding, the structure shows that binding cooperativity and interactions with PAP occur only when U1A is bound to its cognate RNA. This mechanism ensures that the activity of PAP enzyme, which is essential to the cell, is only down regulated when U1A is bound to the U1A mRNA.
Assuntos
Poli A/metabolismo , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/metabolismo , Sequências Reguladoras de Ácido Nucleico/genética , Ribonucleoproteína Nuclear Pequena U1/química , Ribonucleoproteína Nuclear Pequena U1/metabolismo , Regiões 3' não Traduzidas/química , Regiões 3' não Traduzidas/genética , Regiões 3' não Traduzidas/metabolismo , Regulação Alostérica , Sequência de Aminoácidos , Sequência de Bases , Sítios de Ligação , Humanos , Modelos Moleculares , Dados de Sequência Molecular , Peso Molecular , Ressonância Magnética Nuclear Biomolecular , Conformação de Ácido Nucleico , Polinucleotídeo Adenililtransferase/antagonistas & inibidores , Polinucleotídeo Adenililtransferase/metabolismo , Ligação Proteica , Estrutura Secundária de Proteína , Processamento Pós-Transcricional do RNA/genética , RNA Mensageiro/química , RNA Mensageiro/genética , Proteínas de Ligação a RNA/química , Relação Estrutura-Atividade , Especificidade por SubstratoRESUMO
The 3' ends of most eukaryotic pre-mRNAs are generated by 3' endonucleolytic cleavage and subsequent polyadenylation. 3'-end formation can be influenced positively or negatively by various factors. In particular, U1 snRNP acts as an inhibitor when bound to a 5' splice site located either upstream of the 3'-end formation signals of bovine papilloma virus (BPV) late transcripts or downstream of the 3'-end processing signals in the 5' LTR of the HIV-1 provirus. Previous work showed that in BPV it is not the first step, 3' cleavage, that is affected by U1 snRNP, but rather the second step, polyadenylation, that is inhibited. Since in HIV-1 the biological requirement is to produce transcripts that read through the 5' LTR cleavage site rather than being cleaved there, this mechanism seemed unlikely to apply. The obvious difference between the two examples was the relative orientation of the 3'-end formation signals and the U1 snRNP-binding site. In vitro assays were therefore used to assess the effect of U1 snRNP bound at various locations relative to a cleavage/polyadenylation site on the 3' cleavage reaction. U1 snRNP was found to inhibit cleavage when bound to a 5' splice site downstream of the cleavage/polyadenylation site, as in the HIV-1 LTR. U1 snRNP binding at this location was shown not to affect the recruitment of multiple cleavage/polyadenylation factors to the cleavage substrate, indicating that inhibition is unlikely to be due to steric hindrance. Interactions between U1A, U1 70K, and poly(A) polymerase, which mediate the effect of U1 snRNP on polyadenylation of other pre-mRNAs, were shown not to be required for cleavage inhibition. Therefore, U1 snRNP bound to a 5' splice site can inhibit cleavage and polyadenylation in two mechanistically different ways depending on whether the 5' splice site is located upstream or downstream of the cleavage site.
Assuntos
Precursores de RNA/metabolismo , Ribonucleoproteína Nuclear Pequena U1/metabolismo , Animais , Sequência de Bases , Sítios de Ligação , Papillomavirus Bovino 1/genética , Papillomavirus Bovino 1/metabolismo , Bovinos , Repetição Terminal Longa de HIV , HIV-1/genética , HIV-1/metabolismo , Células HeLa , Humanos , Mutação , Polinucleotídeo Adenililtransferase/metabolismo , Precursores de RNA/química , Precursores de RNA/genética , Processamento Pós-Transcricional do RNA , Splicing de RNARESUMO
Although it has been established that the processing factors involved in pre-mRNA splicing and 3'-end formation can influence each other positively, the molecular basis of this coupling interaction was not known. Stimulation of pre-mRNA splicing by an adjacent cis-linked cleavage and polyadenylation site in HeLa cell nuclear extract is shown to occur at an early step in splicing, the binding of U2AF 65 to the pyrimidine tract of the intron 3' splice site. The carboxyl terminus of poly(A) polymerase (PAP) previously has been implicated indirectly in the coupling process. We demonstrate that a fusion protein containing the 20 carboxy-terminal amino acids of PAP, when tethered downstream of an intron, increases splicing efficiency and, like the entire 3'-end formation machinery, stimulates U2AF 65 binding to the intron. The carboxy-terminal domain of PAP makes a direct and specific interaction with residues 17-47 of U2AF 65, implicating this interaction in the coupling of splicing and 3'-end formation.
Assuntos
Regiões 3' não Traduzidas/metabolismo , Proteínas Nucleares , Polinucleotídeo Adenililtransferase/metabolismo , Precursores de RNA/metabolismo , Processamento Pós-Transcricional do RNA/fisiologia , Ribonucleoproteínas/metabolismo , Sequência de Aminoácidos , Células HeLa/metabolismo , Humanos , Íntrons , Substâncias Macromoleculares , Dados de Sequência Molecular , Proteínas de Neoplasias/metabolismo , Ligação Proteica , Splicing de RNA/fisiologia , RNA Neoplásico/metabolismo , Sequências Reguladoras de Ácido Nucleico , Ribonucleoproteínas Nucleares Pequenas/metabolismo , Fator de Processamento U2AFRESUMO
Nuclear import of the two uracil-rich small nuclear ribonucleoprotein (U snRNP) components U1A and U2B" is mediated by unusually long and complex nuclear localization signals (NLSs). Here we investigate nuclear import of U1A and U2B" in vitro and demonstrate that it occurs by an active, saturable process. Several lines of evidence suggest that import of the two proteins occurs by an import mechanism different to those characterized previously. No cross competition is seen with a variety of previously studied NLSs. In contrast to import mediated by members of the importin-beta family of nucleocytoplasmic transport receptors, U1A/U2B" import is not inhibited by either nonhydrolyzable guanosine triphosphate (GTP) analogues or by a mutant of the GTPase Ran that is incapable of GTP hydrolysis. Adenosine triphosphate is capable of supporting U1A and U2B" import, whereas neither nonhydrolyzable adenosine triphosphate analogues nor GTP can do so. U1A and U2B" import in vitro does not require the addition of soluble cytosolic proteins, but a factor or factors required for U1A and U2B" import remains tightly associated with the nuclear fraction of conventionally permeabilized cells. This activity can be solubilized in the presence of elevated MgCl(2). These data suggest that U1A and U2B" import into the nucleus occurs by a hitherto uncharacterized mechanism.
Assuntos
Trifosfato de Adenosina/metabolismo , Proteínas Nucleares/metabolismo , Proteínas de Ligação a RNA , Ribonucleoproteína Nuclear Pequena U1/metabolismo , Ribonucleoproteína Nuclear Pequena U2/metabolismo , Spliceossomos/metabolismo , Sequência de Aminoácidos , Autoantígenos , Transporte Biológico , Compartimento Celular , Permeabilidade da Membrana Celular , Núcleo Celular/metabolismo , Citosol/metabolismo , Guanosina Trifosfato/análogos & derivados , Células HeLa , Humanos , Hidrólise , Dados de Sequência Molecular , Mutação , Ribonucleoproteínas Nucleares Pequenas , Proteína ran de Ligação ao GTP/genética , Proteína ran de Ligação ao GTP/metabolismo , Proteínas Centrais de snRNPRESUMO
Yeast strains lacking the yeast nuclear cap-binding complex (yCBC) are viable, although impaired in growth. We have taken advantage of this observation to carry out a genetic screen for components that show synthetic lethality (SL) with a cbp20-Delta cbp80-Delta double mutation. One set of SL interactions was due to mutations that were complemented by components of U1 small nuclear RNP (snRNP) and the yeast splicing commitment complex. These interactions confirm the role of yCBC in commitment complex formation. Physical interaction of yCBC with the commitment complex components Mud10p and Mud2p, which may directly mediate yCBC function, was demonstrated. Unexpectedly, we identified multiple SL mutations that were complemented by Cbf5p and Nop58p. These are components of the two major classes of yeast small nucleolar RNPs, which function in the maturation of rRNA precursors. Mutants lacking yCBC were found to be defective in rRNA processing. Analysis of the yCBC deletion phenotype suggests that this is likely to be due to a defect in the splicing of a subset of ribosomal protein mRNA precursors.
Assuntos
Proteínas Nucleares/metabolismo , Capuzes de RNA/metabolismo , Splicing de RNA , Proteínas de Ligação a RNA/metabolismo , Saccharomyces cerevisiae/genética , Deleção de Genes , Genes Letais , Teste de Complementação Genética , Proteínas Nucleares/genética , Ligação Proteica , Proteínas de Ligação ao Cap de RNA , Precursores de RNA/metabolismo , RNA Mensageiro/biossíntese , RNA Ribossômico/biossíntese , Proteínas de Ligação a RNA/genéticaRESUMO
The characterization of a novel yeast-splicing factor, Luc7p, is presented. The LUC7 gene was identified by a mutation that causes lethality in a yeast strain lacking the nuclear cap-binding complex (CBC). Luc7p is similar in sequence to metazoan proteins that have arginine-serine and arginine-glutamic acid repeat sequences characteristic of a family of splicing factors. We show that Luc7p is a component of yeast U1 snRNP and is essential for vegetative growth. The composition of yeast U1 snRNP is altered in luc7 mutant strains. Extracts of these strains are unable to support any of the defined steps of splicing unless recombinant Luc7p is added. Although the in vivo defect in splicing wild-type reporter introns in a luc7 mutant strain is comparatively mild, splicing of introns with nonconsensus 5' splice site or branchpoint sequences is more defective in the mutant strain than in wild-type strains. By use of reporters that have two competing 5' splice sites, a loss of efficient splicing to the cap proximal splice site is observed in luc7 cells, analogous to the defect seen in strains lacking CBC. CBC can be coprecipitated with U1 snRNP from wild-type, but not from luc7, yeast strains. These data suggest that the loss of Luc7p disrupts U1 snRNP-CBC interaction, and that this interaction contributes to normal 5' splice site recognition.
Assuntos
Precursores de RNA/metabolismo , Splicing de RNA/fisiologia , Ribonucleoproteína Nuclear Pequena U1/fisiologia , Processamento Alternativo/fisiologia , Sequência de Aminoácidos , Evolução Molecular , Proteínas Fúngicas/genética , Humanos , Modelos Genéticos , Dados de Sequência Molecular , Mutação , Capuzes de RNA/metabolismo , Proteínas de Ligação a RNA/metabolismo , Saccharomyces cerevisiae/genética , Homologia de Sequência de Aminoácidos , Temperatura , Fatores de TempoRESUMO
CRM1 is an export receptor mediating rapid nuclear exit of proteins and RNAs to the cytoplasm. CRM1 export cargoes include proteins with a leucine-rich nuclear export signal (NES) that bind directly to CRM1 in a trimeric complex with RanGTP. Using a quantitative CRM1-NES cargo binding assay, significant differences in affinity for CRM1 among natural NESs are demonstrated, suggesting that the steady-state nucleocytoplasmic distribution of shuttling proteins could be determined by the relative strengths of their NESs. We also show that a trimeric CRM1-NES-RanGTP complex is disassembled by RanBP1 in the presence of RanGAP, even though RanBP1 itself contains a leucine-rich NES. Selection of CRM1-binding proteins from Xenopus egg extract leads to the identification of an NES-containing DEAD-box helicase, An3, that continuously shuttles between the nucleus and the cytoplasm. In addition, we identify the Xenopus homologue of the nucleoporin CAN/Nup214 as a RanGTP- and NES cargo-specific binding site for CRM1, suggesting that this nucleoporin plays a role in export complex disassembly and/or CRM1 recycling.
Assuntos
Proteínas de Transporte/metabolismo , Carioferinas , Complexo de Proteínas Formadoras de Poros Nucleares , Proteínas Nucleares/metabolismo , RNA Helicases/metabolismo , Receptores Citoplasmáticos e Nucleares , Sequência de Aminoácidos , Animais , Sequência de Bases , Transporte Biológico Ativo , Núcleo Celular/metabolismo , Primers do DNA/genética , Feminino , Proteínas de Ligação ao GTP/metabolismo , Humanos , Técnicas In Vitro , Modelos Biológicos , Dados de Sequência Molecular , Oócitos/metabolismo , Ligação Proteica , RNA Helicases/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Homologia de Sequência de Aminoácidos , Xenopus , Proteína ran de Ligação ao GTP , Proteína Exportina 1RESUMO
Chromosomes are segregated by two antiparallel arrays of microtubules arranged to form the spindle apparatus. During cell division, the nucleation of cytosolic microtubules is prevented and spindle microtubules nucleate from centrosomes (in mitotic animal cells) or around chromosomes (in plants and some meiotic cells). The molecular mechanism by which chromosomes induce local microtubule nucleation in the absence of centrosomes is unknown, but it can be studied by adding chromatin beads to Xenopus egg extracts. The beads nucleate microtubules that eventually reorganize into a bipolar spindle. RCC1, the guanine-nucleotide-exchange factor for the GTPase protein Ran, is a component of chromatin. Using the chromatin bead assay, we show here that the activity of chromosome-associated RCC1 protein is required for spindle formation. Ran itself, when in the GTP-bound state (Ran-GTP), induces microtubule nucleation and spindle-like structures in M-phase extract. We propose that RCC1 generates a high local concentration of Ran-GTP around chromatin which in turn induces the local nucleation of microtubules.