RESUMO
Nucleocytoplasmic transport occurs through nuclear pore complexes (NPCs) and is mediated by saturable transport receptors that shuttle between the nucleus and cytoplasm. Our understanding of the molecular interactions underlying this process has improved dramatically as a result of the elucidation of the crystal structures of several nuclear transport factors either alone or in a complex with other components of the nuclear transport machinery. Furthermore, a conserved family of proteins, which is distinct from the well characterized family of importin beta-like nuclear export receptors, is implicated in the export of messenger RNA to the cytoplasm.
Assuntos
Proteínas de Transporte/metabolismo , Sinais de Localização Nuclear/metabolismo , Poro Nuclear/metabolismo , Proteínas Nucleares/metabolismo , Proteínas de Transporte Nucleocitoplasmático , Proteína ran de Ligação ao GTP/metabolismo , Transporte Ativo do Núcleo Celular/fisiologia , Animais , Sítios de Ligação/fisiologia , Núcleo Celular/metabolismo , Citoplasma/metabolismo , Humanos , Carioferinas , Sinais de Localização Nuclear/química , Poro Nuclear/ultraestrutura , Proteínas Nucleares/química , Splicing de RNA , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/metabolismo , Translocação GenéticaRESUMO
Various classes of RNA are exported from the nucleus to the cytoplasm, including transcripts of RNA polymerase I (large ribosomal RNAs), II (U-rich small nuclear RNAs [U snRNAs], mRNAs), and III (tRNAs, 5S RNA). Here, evidence is presented that some steps in the export of various classes of nuclear RNA are mediated by specific rather than common factors. Using microinjection into Xenopus oocytes, it is shown that a tRNA, a U snRNA, and an mRNA competitively inhibit their own export at concentrations at which they have no effect on the export of heterologous RNAs. While the export of both U snRNAs and mRNAs is enhanced by their 7-methyl guanosine cap structures, factors recognizing this structure are found to be limiting in concentration only in the case of U snRNAs. In addition to the specific factors, evidence for steps in the export process that may be common to at least some classes of RNA are provided by experiments in which synthetic homopolymeric RNAs are used as inhibitors.
Assuntos
Núcleo Celular/metabolismo , Oócitos/metabolismo , RNA de Transferência de Metionina/metabolismo , RNA/metabolismo , Animais , Sequência de Bases , Primers do DNA , Feminino , Humanos , Cinética , Microinjeções , Dados de Sequência Molecular , Reação em Cadeia da Polimerase , RNA/administração & dosagem , RNA/biossíntese , RNA Polimerase I/metabolismo , RNA Polimerase II/metabolismo , RNA Mensageiro/metabolismo , RNA Nuclear Pequeno/metabolismo , RNA de Transferência de Metionina/biossíntese , Xenopus laevisRESUMO
It has previously been shown that efficient export of U1 snRNA or of microinjected, in vitro synthesized, RNA transcripts from the nucleus of Xenopus oocytes is facilitated by their monomethyl guanosine cap structures. Nuclear exit of these transcripts could be competitively inhibited by microinjection of an excess of a cap analog, the dinucleotide m7GpppG (Hamm, J., and I. W. Mattaj. 1990. Cell. 63:109-118). We have now analyzed the ability of several other related cap analogs to inhibit the export of U1 snRNA from the nucleus. The results define the recognition specificity of a factor(s) involved in RNA transport, and indicate that the cap binding activity (CBA) involved in RNA export is different from cap binding proteins (CBPs) involved in the initiation of translation. A CBP, whose specificity for different analogs correlates with the ability of the analogs to inhibit U1 snRNA export, is identified in nuclear extracts prepared from HeLa cells. We propose that this protein may have a role in the export of capped RNAs from the nucleus.
Assuntos
Capuzes de RNA/metabolismo , RNA Polimerase II/metabolismo , RNA Nuclear Pequeno/metabolismo , Proteínas de Ligação a RNA/metabolismo , Transcrição Gênica/genética , Transporte Biológico Ativo , Núcleo Celular/metabolismo , Cromatografia , Células HeLa , Humanos , Plasmídeos/genética , Proteínas de Ligação ao Cap de RNARESUMO
In vertebrates, a nuclear cap-binding complex (CBC) formed by two cap- binding proteins, CBP20 and CBP80, is involved in several steps of RNA metabolism, including pre-mRNA splicing and nuclear export of some RNA polymerase II-transcribed U snRNAs. The CBC is highly conserved, and antibodies against human CBP20 cross-react with the CBP20 counterpart in the dipteran Chironomus tentans. Using immunoelectron microscopy, the in situ association of CBP20 with a specific pre-mRNP particle, the Balbiani ring particle, has been analyzed at different stages of pre-mRNA synthesis, maturation, and nucleo-cytoplasmic transport. We demonstrate that CBP20 binds to the nascent pre-mRNA shortly after transcription initiation, stays in the RNP particles after splicing has been completed, and remains attached to the 5' domain during translocation of the RNP through the nuclear pore complex (NPC). The rapid association of CBP20 with nascent RNA transcripts in situ is consistent with the role of CBC in splicing, and the retention of CBC on the RNP during translocation through the NPC supports its proposed involvement in RNA export.
Assuntos
Núcleo Celular/metabolismo , Cromossomos/metabolismo , Precursores de RNA/metabolismo , Proteínas de Ligação a RNA/metabolismo , Ribonucleoproteínas/metabolismo , Animais , Transporte Biológico , Núcleo Celular/química , Chironomidae , Citoplasma/química , Células HeLa , Humanos , Membrana Nuclear/metabolismo , Proteínas de Ligação ao Cap de RNA , Capuzes de RNA/metabolismo , Splicing de RNA , Proteínas de Ligação a RNA/análise , Especificidade da Espécie , Transcrição GênicaRESUMO
Among the nuclear proteins associated with mRNAs before their export to the cytoplasm are the abundant heterogeneous nuclear (hn) RNPs. Several of these contain the M9 signal that, in the case of hnRNP A1, has been shown to be sufficient to signal both nuclear export and nuclear import in cultured somatic cells. Kinetic competition experiments are used here to demonstrate that M9-directed nuclear import in Xenopus oocytes is a saturable process. Saturating levels of M9 have, however, no effect on the import of either U snRNPs or proteins carrying a classical basic NLS. Previous work demonstrated the existence of nuclear export factors specific for particular classes of RNA. Injection of hnRNP A1 but not of a mutant protein lacking the M9 domain inhibited export of mRNA but not of other classes of RNA. This suggests that hnRNP A1 or other proteins containing an M9 domain play a role in mRNA export from the nucleus. However, the requirement for M9 function in mRNA export is not identical to that in hnRNP A1 protein transport.
Assuntos
Núcleo Celular/metabolismo , Ribonucleoproteínas Nucleares Heterogêneas Grupo A-B , Sinais Direcionadores de Proteínas/fisiologia , Ribonucleoproteínas/fisiologia , Animais , Transporte Biológico/fisiologia , Feminino , Ribonucleoproteína Nuclear Heterogênea A1 , Ribonucleoproteínas Nucleares Heterogêneas , Oócitos/fisiologia , Estrutura Terciária de Proteína , Splicing de RNA/fisiologia , RNA Mensageiro/metabolismo , RNA Nuclear Pequeno/metabolismo , Proteínas de Ligação a RNA/química , Proteínas de Ligação a RNA/fisiologia , Ribonucleoproteínas/química , XenopusRESUMO
Importin beta is a major mediator of import into the cell nucleus. Importin beta binds cargo molecules either directly or via two types of adapter molecules, importin alpha, for import of proteins with a classical nuclear localization signal (NLS), or snurportin 1, for import of m3G-capped U snRNPs. Both adapters have an NH2-terminal importin beta-binding domain for binding to, and import by, importin beta, and both need to be returned to the cytoplasm after having delivered their cargoes to the nucleus. We have shown previously that CAS mediates export of importin alpha. Here we show that snurportin 1 is exported by CRM1, the receptor for leucine-rich nuclear export signals (NESs). However, the interaction of CRM1 with snurportin 1 differs from that with previously characterized NESs. First, CRM1 binds snurportin 1 50-fold stronger than the Rev protein and 5,000-fold stronger than the minimum Rev activation domain. Second, snurportin 1 interacts with CRM1 not through a short peptide but rather via a large domain that allows regulation of affinity. Strikingly, snurportin 1 has a low affinity for CRM1 when bound to its m3G-capped import substrate, and a high affinity when substrate-free. This mechanism appears crucial for productive import cycles as it can ensure that CRM1 only exports snurportin 1 that has already released its import substrate in the nucleus.
Assuntos
Proteínas de Transporte/metabolismo , Receptores Citoplasmáticos e Nucleares/metabolismo , Ribonucleoproteínas Nucleares Pequenas/metabolismo , Fatores de Transcrição/metabolismo , Animais , Sítios de Ligação , Núcleo Celular/metabolismo , Cromatografia de Afinidade , Citoplasma/metabolismo , Escherichia coli , Feminino , Células HeLa , Humanos , Carioferinas , Cinética , Modelos Biológicos , Proteínas Nucleares/isolamento & purificação , Proteínas Nucleares/metabolismo , Oócitos/fisiologia , Proteínas de Ligação ao Cap de RNA , Receptores Citoplasmáticos e Nucleares/isolamento & purificação , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Transdução de Sinais , Xenopus laevis , Proteína Exportina 1RESUMO
The importin-alpha/beta complex and the GTPase Ran mediate nuclear import of proteins with a classical nuclear localization signal. Although Ran has been implicated also in a variety of other processes, such as cell cycle progression, a direct function of Ran has so far only been demonstrated for importin-mediated nuclear import. We have now identified an entire class of approximately 20 potential Ran targets that share a sequence motif related to the Ran-binding site of importin-beta. We have confirmed specific RanGTP binding for some of them, namely for two novel factors, RanBP7 and RanBP8, for CAS, Pse1p, and Msn5p, and for the cell cycle regulator Cse1p from Saccharomyces cerevisiae. We have studied RanBP7 in more detail. Similar to importin-beta, it prevents the activation of Ran's GTPase by RanGAP1 and inhibits nucleotide exchange on RanGTP. RanBP7 binds directly to nuclear pore complexes where it competes for binding sites with importin-beta, transportin, and apparently also with the mediators of mRNA and U snRNA export. Furthermore, we provide evidence for a Ran-dependent transport cycle of RanBP7 and demonstrate that RanBP7 can cross the nuclear envelope rapidly and in both directions. On the basis of these results, we propose that RanBP7 might represent a nuclear transport factor that carries an as yet unknown cargo, which could apply as well for this entire class of related RanGTP-binding proteins.
Assuntos
GTP Fosfo-Hidrolases/metabolismo , Proteínas de Ligação ao GTP/genética , Proteínas Nucleares/genética , Proteína ran de Ligação ao GTP , Sequência de Aminoácidos , Animais , Sítios de Ligação , Transporte Biológico , Núcleo Celular/metabolismo , Sequência Conservada , Citoplasma/metabolismo , DNA Complementar , Proteínas de Ligação ao GTP/metabolismo , Guanosina Trifosfato/metabolismo , Células HeLa , Humanos , Carioferinas , Dados de Sequência Molecular , Membrana Nuclear/metabolismo , Sinais de Localização Nuclear , Proteínas Nucleares/metabolismo , Proteínas/metabolismo , Coelhos , Receptores Citoplasmáticos e Nucleares , Homologia de Sequência de Aminoácidos , Xenopus , beta CarioferinasRESUMO
BACKGROUND: A hallmark of retroviral gene expression is that unspliced retroviral genomic RNA is exported to the cytoplasm, whereas endogenous intron-containing cellular RNAs are usually retained in the nucleus. In complex retroviruses, such as human immunodeficiency virus-1 (HIV-1), nuclear export is accomplished by the interaction of a virally encoded protein, Rev, with a cis-acting RNA element, the Rev-responsive element (RRE). In type D retroviruses, such as the simian retrovirus type 1 (SRV-1), however, genomic RNA is exported by cellular factor(s) that interact with a conserved cis-acting RNA element, the constitutive transport element (CTE). RESULTS: We found that the CTE was exported in a specific and saturable fashion from Xenopus oocyte nuclei. When inserted into the intron of an adenovirus-derived pre-mRNA, the CTE did not affect splicing efficiency but promoted the nuclear export of the excised intron lariat that is normally retained within the nucleus. Export of CTE-containing RNAs to the cytoplasm was not affected by the heterogeneous nuclear ribonucleoprotein A1 or an excess of peptides corresponding to the Rev nuclear export signal. Microinjection of saturating amounts of CTE RNA did not affect tRNA export or Rev-mediated export but did inhibit mRNA export. CTE-mediated export was found to be dependent on Ran-mediated GTP hydrolysis. CONCLUSION: The Rev-RRE system and the CTE direct intron-containing RNAs to distinct export pathways. Although previous data have suggested that Rev uses the same export pathway as uracil-rich small nuclear RNAs and 5S ribosomal RNA, the CTE seems to interact with evolutionarily conserved factors that are essential for cellular mRNA export.
Assuntos
Produtos do Gene rev/metabolismo , HIV-1/genética , Ribonucleoproteínas Nucleares Heterogêneas Grupo A-B , RNA Mensageiro/metabolismo , RNA Viral/metabolismo , Retrovirus dos Símios/genética , Animais , Transporte Biológico , Núcleo Celular/virologia , GTP Fosfo-Hidrolases/metabolismo , Proteínas de Ligação ao GTP/metabolismo , Produtos do Gene rev/genética , Ribonucleoproteína Nuclear Heterogênea A1 , Ribonucleoproteínas Nucleares Heterogêneas , Humanos , Íntrons , Proteínas Nucleares/metabolismo , Oócitos/metabolismo , Splicing de RNA , Ribonucleoproteínas/metabolismo , Soroalbumina Bovina/metabolismo , Xenopus , Proteína ran de Ligação ao GTP , Produtos do Gene rev do Vírus da Imunodeficiência HumanaRESUMO
BACKGROUND: Although X-linked mental retardation (XLMR) affects 2%-3% of the human population, little is known about the underlying molecular mechanisms. Recent interest in this topic led to the identification of several genes for which mutations result in the disturbance of cognitive development. RESULTS: We identified a novel gene that is interrupted by an inv(X)(p21.1;q22) in a male patient with a syndromic form of mental retardation. Molecular analysis of both breakpoint regions did not reveal an interrupted gene on Xp, but identified a novel nuclear RNA export factor (NXF) gene cluster, Xcen-NXF5-NXF2-NXF4-NXF3-Xqter, in which NXF5 is split by the breakpoint, leading to its functional nullisomy. The predicted NXF5 protein shows high similarity with the central part of the presumed mRNA nuclear export factor TAP/NXF1. Functional analysis of NXF5 demonstrates binding to RNA as well as to the RNA nuclear export-associated protein p15/NXT. In contrast to TAP/NXF1, overexpression studies localized NXF5 in the form of granules in the cell body and neurites of mature hippocampal neurons, suggesting a role in mRNA transport. The two newly identified mouse nxf homologs, nxf-a and nxf-b, which also map on X, show highest mRNA levels in the brain. CONCLUSIONS: A novel member of the nuclear RNA export factor family is absent in a male patient with a syndromic form of mental retardation. Although we did not find direct evidence for the involvement of NXF5 in MR, the gene could be involved in development, possibly through a process in mRNA metabolism in neurons.
Assuntos
Deleção de Genes , Deficiência Intelectual/genética , Proteínas Nucleares/genética , Proteínas de Transporte Nucleocitoplasmático , Proteínas de Ligação a RNA/genética , Cromossomo X , Transporte Ativo do Núcleo Celular , Sequência de Aminoácidos , Animais , Encéfalo/metabolismo , Inversão Cromossômica , Clonagem Molecular , Citoplasma/metabolismo , Expressão Gênica , Hipocampo/citologia , Hipocampo/metabolismo , Humanos , Deficiência Intelectual/metabolismo , Masculino , Camundongos , Pessoa de Meia-Idade , Dados de Sequência Molecular , Família Multigênica , Proteínas Nucleares/química , Proteínas Nucleares/metabolismo , Proteínas Nucleares/fisiologia , RNA/metabolismo , Proteínas de Ligação a RNA/química , Proteínas de Ligação a RNA/metabolismo , Proteínas de Ligação a RNA/fisiologia , Homologia de Sequência de Aminoácidos , SíndromeRESUMO
Dbp5 is the only member of the DExH/D box family of RNA helicases that is directly implicated in the export of messenger RNAs from the nucleus of yeast and vertebrate cells. Dbp5 localizes in the cytoplasm and at the cytoplasmic face of the nuclear pore complex (NPC). In an attempt to identify proteins present in a highly enriched NPC fraction, two other helicases were detected: RNA helicase A (RHA) and UAP56. This suggested a role for these proteins in nuclear transport. Contrary to expectation, we show that the Drosophila homolog of Dbp5 is not essential for mRNA export in cultured Schneider cells. In contrast, depletion of HEL, the Drosophila homolog of UAP56, inhibits growth and results in a robust accumulation of polyadenylated RNAs within the nucleus. Consequently, incorporation of [35S]methionine into newly synthesized proteins is inhibited. This inhibition affects the expression of both heat-shock and non-heat-shock mRNAs, as well as intron-containing and intronless mRNAs. In HeLa nuclear extracts, UAP56 preferentially, but not exclusively, associates with spliced mRNAs carrying the exon junction complex (EJC). We conclude that HEL is essential for the export of bulk mRNA in Drosophila. The association of human UAP56 with spliced mRNAs suggests that this protein might provide a functional link between splicing and export.
Assuntos
Adenosina Trifosfatases/metabolismo , Núcleo Celular/metabolismo , Proteínas de Drosophila/metabolismo , RNA Helicases/metabolismo , RNA Mensageiro/metabolismo , Transporte Ativo do Núcleo Celular , Adenosina Trifosfatases/genética , Animais , Células Cultivadas , Clonagem Molecular , RNA Helicases DEAD-box , DNA Complementar , Drosophila melanogaster , Células HeLa , Resposta ao Choque Térmico , Humanos , Biossíntese de Proteínas , RNA Helicases/genética , Splicing de RNA , RNA Antissenso , RNA Interferente PequenoRESUMO
hnRNP F was identified in a screen for proteins that interact with human CBP80 and CBP20, the components of the nuclear cap-binding complex (CBC). In vitro interaction studies showed that hnRNP F can bind to both CBP20 and CBP80 individually. hnRNP F and CBC bind independently to RNA, but hnRNP F binds preferentially to CBC-RNA complexes rather than to naked RNA. The hnRNP H protein, which is 78% identical to hnRNP F and also interacts with both CBP80 and CBP20 in vitro, does not discriminate between naked RNA and CBC-RNA complexes, showing that this effect is specific. Depletion of hnRNP F from HeLa cell nuclear extract decreases the efficiency of pre-mRNA splicing, a defect which can be partially compensated by addition of recombinant hnRNP F. Thus, hnRNP F is required for efficient pre-mRNA splicing in vitro and may participate in the effect of CBC on pre-mRNA splicing.
Assuntos
Reagentes de Ligações Cruzadas/metabolismo , Capuzes de RNA/metabolismo , RNA Nuclear Heterogêneo/metabolismo , Proteínas de Ligação a RNA/metabolismo , Ribonucleoproteínas/metabolismo , Sequência de Aminoácidos , Eletroforese em Gel de Poliacrilamida , Células HeLa , Ribonucleoproteínas Nucleares Heterogêneas Grupo F-H , Ribonucleoproteínas Nucleares Heterogêneas , Humanos , Substâncias Macromoleculares , Dados de Sequência Molecular , Proteínas de Ligação ao Cap de RNARESUMO
The human immunodeficiency virus type 1 Rev protein contains a nuclear export signal (NES) that is required for Rev-mediated RNA export in mammals as well as in the yeast Saccharomyces cerevisiae. The Rev NES has been shown to specifically interact with a human (hRIP/RAB1) and a yeast (yRip1p) protein in the two-hybrid assay. Both of these interacting proteins are related to FG nucleoporins on the basis of the presence of typical repeat motifs. This paper shows that Rev is able to interact with multiple FG repeat-containing nucleoporins from both S. cerevisiae and mammals; moreover, the ability of Rev NES mutants to interact with these FG nucleoporins parallels the ability of the mutants to promote RNA export in yeast and mammalian cells. The data also show that, after Xenopus oocyte nuclear injection, several FG nucleoporin repeat domains inhibit the export of both Rev protein and U small nuclear RNAs, suggesting that these nucleoporins participate in Rev-mediated and cellular RNA export. Interestingly, not all FG nucleoporin repeat domains produced the same pattern of RNA export inhibition. The results suggest that Rev and cellular mediators of RNA export can interact with multiple components of the nuclear pore complex during transport, analogous to the proposed mode of action of the nuclear protein import receptor.
Assuntos
Regulação da Expressão Gênica , Produtos do Gene rev/metabolismo , HIV-1/genética , Proteínas Nucleares/genética , RNA Viral/metabolismo , Saccharomyces cerevisiae/genética , Transporte Biológico/genética , Núcleo Celular/metabolismo , Produtos do Gene rev/genética , HIV-1/metabolismo , Humanos , Proteínas Nucleares/metabolismo , RNA Viral/genética , Sequências Repetitivas de Ácido Nucleico , Saccharomyces cerevisiae/metabolismo , Produtos do Gene rev do Vírus da Imunodeficiência HumanaRESUMO
The human immunodeficiency virus type 1 (HIV) Rev protein is thought to be involved in the export of unspliced or singly spliced viral mRNAs from the nucleus to the cytoplasm. This function is mediated by a sequence-specific interaction with a cis-acting RNA element, the Rev response element (RRE), present in these intron-containing RNAs. To identify possible host proteins involved in Rev function, we fractionated nuclear cell extracts with a Rev affinity column. A single, tightly associated Rev-binding protein was identified; this protein is the mammalian nucleolar protein B23. The interaction between HIV Rev and B23 is very specific, as it was observed in complex cell extracts. The complex is also very stable toward dissociation by high salt concentrations. Despite the stability of the Rev-B23 protein complex, the addition of RRE, but not control RNA, led to the displacement of B23 and the formation of a specific Rev-RRE complex. The mammalian nucleolar protein B23 or its amphibian counterpart No38 is believed to function as a shuttle receptor for the nuclear import of ribosomal proteins. B23 may also serve as a shuttle for the import of HIV Rev from the cytoplasm into the nucleus or nucleolus to allow further rounds of export of RRE-containing viral RNAs.
Assuntos
Produtos do Gene rev/metabolismo , Genes rev , HIV-1/metabolismo , Proteínas Nucleares/metabolismo , Sítios de Ligação , Núcleo Celular/metabolismo , Centrifugação com Gradiente de Concentração , Cromatografia de Afinidade , DNA Viral/genética , DNA Viral/isolamento & purificação , Produtos do Gene rev/isolamento & purificação , HIV-1/genética , Células HeLa/metabolismo , Humanos , Proteínas Nucleares/isolamento & purificação , Nucleofosmina , Plasmídeos , Ligação Proteica , Transcrição Gênica , Produtos do Gene rev do Vírus da Imunodeficiência HumanaRESUMO
Vertebrate TAP (also called NXF1) and its yeast orthologue, Mex67p, have been implicated in the export of mRNAs from the nucleus. The TAP protein includes a noncanonical RNP-type RNA binding domain, four leucine-rich repeats, an NTF2-like domain that allows heterodimerization with p15 (also called NXT1), and a ubiquitin-associated domain that mediates the interaction with nucleoporins. Here we show that TAP belongs to an evolutionarily conserved family of proteins that has more than one member in higher eukaryotes. Not only the overall domain organization but also residues important for p15 and nucleoporin interaction are conserved in most family members. We characterize two of four human TAP homologues and show that one of them, NXF2, binds RNA, localizes to the nuclear envelope, and exhibits RNA export activity. NXF3, which does not bind RNA or localize to the nuclear rim, has no RNA export activity. Database searches revealed that although only one p15 (nxt) gene is present in the Drosophila melanogaster and Caenorhabditis elegans genomes, there is at least one additional p15 homologue (p15-2 [also called NXT2]) encoded by the human genome. Both human p15 homologues bind TAP, NXF2, and NXF3. Together, our results indicate that the TAP-p15 mRNA export pathway has diversified in higher eukaryotes compared to yeast, perhaps reflecting a greater substrate complexity.
Assuntos
Transporte Ativo do Núcleo Celular , Proteínas Nucleares/genética , Proteínas de Transporte Nucleocitoplasmático , Proteínas de Ligação a RNA/genética , Sequência de Aminoácidos , Animais , Sítios de Ligação , Caenorhabditis elegans , Proteínas de Caenorhabditis elegans , Proteínas de Transporte/metabolismo , Sequência Conservada , Dimerização , Proteínas de Drosophila , Drosophila melanogaster , Evolução Molecular , Duplicação Gênica , Humanos , Dados de Sequência Molecular , Família Multigênica , Poro Nuclear/metabolismo , Proteínas Nucleares/metabolismo , Homologia de Sequência de AminoácidosRESUMO
Scaffold-associated regions (SARs) are A + T-rich DNA regions of several hundred base-pairs that are known to bind specifically to nuclear or metaphase scaffolds. Surprisingly, histone H1 specifically associates with SARs. Under conditions of high co-operativity, at input ratios of H1 to DNA up to 15% (w/w), histone H1 binds preferentially to those DNA molecules harboring a SAR, leaving the non-SAR fragments free. Our experiments identify SARs as cis-acting sequences that nucleate co-operative H1 assembly along the SAR into the flanking non-SAR DNA. Experiments with simple DNA polymers implicate homopolymeric oligo(dA).oligo(dT) tracts in preferential histone H1 assembly. The homopolymer oligo(dA).oligo(dT) is, above a critical length of 130 base-pairs, a highly specific nucleator of H1 assembly. SARs may control the conformation of chromatin domains via a regulated H1 assembly and set up the potential transcriptional repertoire of the cell.
Assuntos
Cromatina/ultraestrutura , DNA/metabolismo , Histonas/metabolismo , Proteínas Nucleares/metabolismo , Animais , Sequência de Bases , Clonagem Molecular , Desoxirribonucleoproteínas/ultraestrutura , Técnicas In Vitro , Dados de Sequência Molecular , Morfogênese , Plasmídeos , Ratos , Sequências Reguladoras de Ácido NucleicoRESUMO
Scaffold-associated regions (SARs) are A + T-rich sequences defined by their specific interaction with the nuclear scaffold. These sequences also direct highly specific binding to purified histone H1, and are characterized by the presence of oligo(dA).oligo(dT) tracts, which are a target for the drug distamyin, an antibiotic with a wide range of biological activities. The interaction of distamycin with SAR sequences results in the complete suppression of binding to either scaffolds or histone H1, suggesting that (dA.dT)n tracts play a direct role in mediating these specific interactions and that histone H1 and nuclear scaffold proteins may recognize a characteristic minor groove width or conformation. The effect of distamycin on these specific DNA-protein interactions in vitro suggests that binding of SARs to the nuclear scaffold and SAR-dependent nucleation of H1 assembly might be important targets of the drug in vivo.
Assuntos
Cromatina/ultraestrutura , DNA/metabolismo , Distamicinas/farmacologia , Histonas/metabolismo , Oligodesoxirribonucleotídeos/metabolismo , Poli dA-dT/metabolismo , Polidesoxirribonucleotídeos/metabolismo , Pirróis/farmacologia , Animais , Núcleo Celular/metabolismo , Núcleo Celular/ultraestrutura , Sistema Livre de Células , Histonas/genética , Técnicas In Vitro , Morfogênese , Ratos , Sequências Reguladoras de Ácido Nucleico , Mapeamento por RestriçãoRESUMO
We have previously identified a number of specific DNA fragments called SARs (scaffold-associated regions) that are associated with the nuclear scaffold and define the basis of DNA loops. We demonstrate that cloned DNA fragments containing SAR sequences bind to nuclear scaffolds in vitro with the same specificity as have genomic SAR fragments. This specific interaction is observed with the biochemically complex type I scaffolds. These scaffolds are composed of the nuclear lamina proteins and a set of other proteins that forms the internal network of these structures. So-called type II scaffolds, which are composed primarily of the lamina proteins and lack the proteins of the internal network, do not bind the SAR fragments at a detectable level. Competition experiments show that different SARs share common structural elements and can bind to the same sites on the nuclear scaffold, although with different affinities. Moreover, the SAR binding sites appear to be evolutionarily conserved, as all the Drosophila SARs also bind with identical specificity to nuclear scaffolds derived from rat liver nuclei. These Sar interaction studies were carried out with lithium 3,5-diiodosalicylate-extracted nuclei. Interestingly, scaffolds prepared by high-salt extraction also bind the genomic and exogenously added SAR fragments specifically. However, the endogenous transcribed sequences, as opposed to the same fragments added as purified DNA, associate randomly with these scaffolds.
Assuntos
DNA/metabolismo , Proteínas Nucleares/metabolismo , Animais , Sítios de Ligação , Evolução Biológica , Mapeamento Cromossômico , Drosophila melanogaster , Técnicas In Vitro , Conformação de Ácido NucleicoRESUMO
microRNAs (miRNAs) represent a novel class of genome-encoded eukaryotic regulatory RNAs that silence gene expression posttranscriptionally. Although the proteins mediating miRNA biogenesis and function have been identified, the precise mechanism by which miRNAs regulate the expression of target mRNAs remains unclear. We summarize recent work from our laboratory demonstrating that miRNAs silence gene expression by at least two independent mechanisms: by repressing translation and/or by promoting mRNA degradation. In Drosophila, both mechanisms require Argonaute 1 (AGO1) and the P-body component GW182. Moreover, mRNA degradation by miRNAs is effected by the enzymes involved in general mRNA decay, including deadenylases and decapping enzymes, which also localize to P bodies. Our findings suggest a model for miRNA function in which AGO1 associates with miRNA targets through miRNA:mRNA base-pairing interactions. GW182 interacts with AGO1 and recruits deadenylases and decapping enzymes, leading to mRNA degradation. However, not all miRNA targets are degraded: Some stay in a translationally silent state, from which they may eventually be released. We propose that the final outcome of miRNA regulation (i.e., degradation vs. translational repression) is influenced by other RNA-binding proteins interacting with the targeted mRNA.
Assuntos
Inativação Gênica , MicroRNAs/genética , MicroRNAs/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Animais , Proteínas Argonautas , Drosophila/genética , Drosophila/metabolismo , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Fatores de Iniciação em Eucariotos , Modelos Biológicos , Biossíntese de Proteínas , Capuzes de RNA/genética , Capuzes de RNA/metabolismo , Estabilidade de RNARESUMO
The distinguishing feature of eukaryotic cells is the segregation of RNA biogenesis and DNA replication in the nucleus, separate from the cytoplasmic machinery for protein synthesis. As a consequence, messenger RNAs (mRNAs) and all cytoplasmic RNAs from nuclear origin need to be transported from their site of synthesis in the nucleus to their final cytoplasmic destination. Nuclear export occurs through nuclear pore complexes (NPCs) and is mediated by saturable transport receptors, which shuttle between the nucleus and cytoplasm. The past years have seen great progress in the characterization of the mRNA export pathway and the identification of proteins involved in this process. A novel family of nuclear export receptors (the NXF family), distinct from the well-characterized family of importin beta-like proteins, has been implicated in the export of mRNA to the cytoplasm.