RESUMO
The yeast Sac3:Cdc31:Sus1:Thp1 (TREX-2) complex facilitates the repositioning and association of actively transcribing genes with nuclear pores (NPCs)-"gene gating"-that is central to integrating transcription, processing, and mRNA nuclear export. We present here the crystal structure of Sus1 and Cdc31 bound to a central region of Sac3 (the CID domain) that is crucial for its function. Sac3(CID) forms a long, gently undulating alpha helix around which one Cdc31 and two Sus1 chains are wrapped. Sus1 has an articulated helical hairpin fold that facilitates its wrapping around Sac3. In vivo studies using engineered mutations that selectively disrupted binding of individual chains to Sac3 indicated that Sus1 and Cdc31 function synergistically to promote NPC association of TREX-2 and mRNA nuclear export. These data indicate Sac3(CID) provides a scaffold within TREX-2 to integrate interactions between protein complexes to facilitate the coupling of transcription and mRNA export during gene expression.
Assuntos
Proteínas de Ligação ao Cálcio/química , Proteínas de Ciclo Celular/química , Poro Nuclear/metabolismo , Proteínas Nucleares/química , Transporte de RNA , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/química , Proteínas de Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/fisiologia , Sequência de Aminoácidos , Proteínas de Ligação ao Cálcio/genética , Proteínas de Ligação ao Cálcio/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Cristalografia por Raios X , Hibridização In Situ , Microscopia de Fluorescência , Dados de Sequência Molecular , Poro Nuclear/genética , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Proteínas de Transporte Nucleocitoplasmático , Porinas , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Homologia de Sequência de AminoácidosRESUMO
The conserved Sac3:Thp1:Sem1:Sus1:Cdc31 (TREX2) complex binds to nuclear pore complexes (NPCs) and, in addition to integrating mRNA nuclear export with preceding steps in the gene expression pathway, facilitates re-positioning of highly regulated actively transcribing genes (such as GAL1) to NPCs. Although TREX2 is thought to bind NPC protein Nup1, defining the precise role of this interaction has been frustrated by the complex pleiotropic phenotype exhibited by nup1Δ strains. To provide a structural framework for understanding the binding of TREX2 to NPCs and its function in the gene expression pathway, we have determined the structure of the Nup1:TREX2 interaction interface and used this information to engineer a Sac3 variant that impairs NPC binding while not compromising TREX2 assembly. This variant inhibited the NPC association of both de-repressed and activated GAL1 and also produced mRNA export and growth defects. These results indicate that the TREX2:Nup1 interaction facilitates the efficient nuclear export of bulk mRNA together with the re-positioning of GAL1 to NPCs that is required for transcriptional control that is mediated by removal of SUMO from repressors by NPC-bound Ulp1.
Assuntos
Galactoquinase/genética , Complexo de Proteínas Formadoras de Poros Nucleares/química , Poro Nuclear/metabolismo , Proteínas de Transporte Nucleocitoplasmático/química , Porinas/química , RNA Mensageiro/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Transporte Ativo do Núcleo Celular , Núcleo Celular/metabolismo , Modelos Moleculares , Mutação , Complexo de Proteínas Formadoras de Poros Nucleares/metabolismo , Proteínas Nucleares/química , Proteínas Nucleares/metabolismo , Proteínas de Transporte Nucleocitoplasmático/genética , Proteínas de Transporte Nucleocitoplasmático/metabolismo , Porinas/genética , Porinas/metabolismo , Domínios e Motivos de Interação entre Proteínas , Transporte de RNA , Proteínas de Ligação a RNA/química , Proteínas de Ligação a RNA/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crescimento & desenvolvimento , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMO
Export of messenger RNA (mRNA) from the nucleus to the cytoplasm is a critical step in the gene expression pathway of eukaryotic cells. Here, we report the functional and structural characterization of the mammalian TREX-2 complex and show how it links transcription/processing with nuclear mRNA export. Mammalian TREX-2 is based on a germinal-centre associated nuclear protein (GANP) scaffold to which ENY2, PCID2 and centrins bind and depletion of any of these components inhibits mRNA export. The crystal structure of the GANP:ENY2 complex shows that two ENY2 chains interact directly with GANP, but they have different orientations from those observed on yeast Sac3. GANP is required to recruit ENY2 to nuclear pore complexes (NPCs), but ENY2 is not necessary to recruit GANP, which requires both its CID and MCM3AP domains, together with nucleoporin Nup153. GANP and ENY2 associate with RNA polymerase II and inhibition of mRNA processing redistributes GANP from NPCs into nuclear foci indicating that mammalian TREX-2 is associated with transcription. Thus, we implicate TREX-2 as an integral component of the mammalian mRNA export machinery where it links transcription and nuclear export by facilitating the transfer of mature mRNPs from the nuclear interior to NPCs.
Assuntos
Acetiltransferases/química , Núcleo Celular/genética , Proteínas Nucleares/química , Proteínas Nucleares/metabolismo , RNA Mensageiro/metabolismo , Transcrição Gênica , Acetiltransferases/análise , Acetiltransferases/metabolismo , Transporte Ativo do Núcleo Celular , Sequência de Aminoácidos , Núcleo Celular/metabolismo , Cristalografia por Raios X , Exodesoxirribonucleases/metabolismo , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Modelos Moleculares , Dados de Sequência Molecular , Complexo de Proteínas Formadoras de Poros Nucleares/metabolismo , Proteínas Nucleares/análise , Fosfoproteínas/metabolismo , Transporte de RNA , Fatores de Transcrição/química , Fatores de Transcrição/metabolismoRESUMO
Most of the individual components of the nuclear elements of the gene expression pathway have been identified and high-resolution structural information is becoming available for many of them. Information is also starting to become available on the larger complexes they form and is beginning to give clues about how the dynamics of their interactions generate function. Although the translocation of export-competent messenger ribonucleoprotein particles (mRNPs) through the nuclear pore transport channel that is mediated by interactions with nuclear pore proteins (nucleoporins) is relatively well understood, the precise molecular mechanisms underlying the assembly of export-competent mRNPs in the nucleus and their Dbp5-mediated disassembly in the cytoplasm is less well defined. Considerable information has been obtained on the structure of Dbp5 in its different nucleotide-bound states and in complex with Gle1 or Nup159/NUP214. Although the precise manner by which the Dbp5 ATPase cycle is coupled to mRNP remodelling remains to be established, current models capture many key details of this process. The formation of export-competent mRNPs in the nucleus remains an elusive component of this pathway and the precise nature of the remodelling that generates these mRNPs as well as detailed understanding of the molecular mechanisms by which this step is integrated with the transcriptional, splicing and polyadenylation machinery by the TREX and TREX-2 complexes remain obscure. This article is part of a Special Issue entitled: Nuclear Transport and RNA Processing.
Assuntos
Transporte Ativo do Núcleo Celular , Poro Nuclear , Transporte de RNA , RNA Mensageiro , Núcleo Celular/genética , Núcleo Celular/metabolismo , Citoplasma/genética , Citoplasma/metabolismo , Humanos , Poro Nuclear/química , Poro Nuclear/genética , Poro Nuclear/metabolismo , Complexo de Proteínas Formadoras de Poros Nucleares/genética , Complexo de Proteínas Formadoras de Poros Nucleares/metabolismo , RNA Mensageiro/biossíntese , RNA Mensageiro/química , RNA Mensageiro/genética , Ribonucleoproteínas/genética , Ribonucleoproteínas/metabolismo , Saccharomyces cerevisiaeRESUMO
Sus1 is a central component of the yeast gene gating machinery, the process by which actively transcribing genes such as GAL1 become associated with nuclear pore complexes. Sus1 is a component of both the SAGA transcriptional co-activator complex and the TREX-2 complex that binds to nuclear pore complexes. TREX-2 contains two Sus1 chains that have an articulated helical hairpin fold, enabling them to wrap around an extended alpha-helix in Sac3, following a helical hydrophobic stripe. In SAGA, Sus1 binds to Sgf11 and has been proposed to provide a link between SAGA and TREX-2. We present here the crystal structure of the complex between Sus1 and the N-terminal region of Sgf11 that forms an extended alpha-helix around which Sus1 wraps in a manner that shares some similarities with the Sus1-Sac3 interface in TREX-2. However, the Sus1-binding site on Sgf11 is somewhat shorter than on Sac3 and is based on a narrower hydrophobic stripe. Engineered mutants that disrupt the Sgf11-Sus1 interaction in vitro confirm the importance of the hydrophobic helical stripe in molecular recognition. Helix alpha1 of the Sus1-articulated hairpin does not bind directly to Sgf11 and adopts a wide range of conformations within and between crystal forms, consistent with the presence of a flexible hinge and also with results from previous extensive mutagenesis studies (Klöckner, C., Schneider, M., Lutz, S., Jani, D., Kressler, D., Stewart, M., Hurt, E., and Köhler, A. (2009) J. Biol. Chem. 284, 12049-12056). A single Sus1 molecule cannot bind Sgf11 and Sac3 simultaneously and this, combined with the structure of the Sus1-Sgf11 complex, indicates that Sus1 forms separate subcomplexes within SAGA and TREX-2.
Assuntos
Proteínas Nucleares/química , Proteínas de Ligação a RNA/química , Proteínas de Saccharomyces cerevisiae/química , Transativadores/química , Fatores de Transcrição/química , Acetiltransferases/química , Acetiltransferases/genética , Acetiltransferases/metabolismo , Sequência de Aminoácidos , Sítios de Ligação , Ligação Competitiva , Cristalografia por Raios X , Eletroforese em Gel de Poliacrilamida , Exodesoxirribonucleases/química , Exodesoxirribonucleases/genética , Exodesoxirribonucleases/metabolismo , Interações Hidrofóbicas e Hidrofílicas , Modelos Moleculares , Dados de Sequência Molecular , Mutação , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Proteínas de Transporte Nucleocitoplasmático/genética , Proteínas de Transporte Nucleocitoplasmático/metabolismo , Porinas/genética , Porinas/metabolismo , Ligação Proteica , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Homologia de Sequência de Aminoácidos , Relação Estrutura-Atividade , Transativadores/genética , Transativadores/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
Sus1 is an evolutionary conserved protein that functions both in transcription and mRNA export and has been proposed to contribute to coupling these processes in yeast. Sus1 mediates its different roles as a component of both the histone H2B deubiquitinating module (Sus1-Sgf11-Ubp8-Sgf73) of the SAGA (Spt-Ada-Gcn5 acetyltransferase) transcriptional co-activator and the mRNA export complex, TREX-2 (Sus1-Sac3-Thp1-Cdc31). We have dissected the different functions of Sus1 with respect to its partitioning in transcription and export complexes using a mutational approach. Here we show that the sus1-10 (E18A, S19A, and G20A) and sus1-12 (V73A and D75A) alleles of Sus1 can be dissociated from TREX-2 while leaving its interaction with SAGA largely intact. Conversely, the binding to both TREX-2 and SAGA was impaired in the sus1-11 allele (G37A and W38A), in which two highly conserved residues were mutated. In vitro experiments demonstrated that dissociation of mutant Sus1 from its partners is caused by a reduced affinity toward the TREX-2 subunit, Sac3, and the SAGA factor, Sgf11, respectively. Consistent with the biochemical data, these sus1 mutant alleles showed differential genetic relationships with SAGA and mRNA export mutants. In vivo, all three sus1 mutants were impaired in targeting TREX-2 (i.e. Sac3) to the nuclear pore complexes and exhibited nuclear mRNA export defects. This study has implications for how Sus1, in combination with distinct interaction partners, can regulate diverse aspects of gene expression.