Dataset Information

Crystal structure of the human ATP-dependent splicing and export factor UAP56.

ABSTRACT: Pre-mRNA splicing requires the function of a number of RNA-dependent ATPases/helicases, yet no three-dimensional structure of any spliceosomal ATPases/helicases is known. The highly conserved DECD-box protein UAP56/Sub2 is an essential splicing factor that is also important for mRNA export. The expected ATPase/helicase activity appears to be essential for the UAP56/Sub2 functions. Here, we show that purified human UAP56 is an active RNA-dependent ATPase, and we also report the crystal structures of UAP56 alone and in complex with ADP, as well as a DECD to DEAD mutant. The structures reveal a unique spatial arrangement of the two conserved helicase domains, and ADP-binding induces significant conformational changes of key residues in the ATP-binding pocket. Our structural analyses suggest a specific protein-RNA displacement model of UAP56/Sub2. The detailed structural information provides important mechanistic insights into the splicing function of UAP56/Sub2. The structures also will be useful for the analysis of other spliceosomal DExD-box ATPases/helicases.

SUBMITTER: Shi H

PROVIDER: S-EPMC539749 | biostudies-literature | 2004 Dec

REPOSITORIES: biostudies-literature

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Crystal structure of the human ATP-dependent splicing and export factor UAP56.

Shi Hang H Cordin Olivier O Minder C Michael CM Linder Patrick P Xu Rui-Ming RM

Proceedings of the National Academy of Sciences of the United States of America 20041207 51

Pre-mRNA splicing requires the function of a number of RNA-dependent ATPases/helicases, yet no three-dimensional structure of any spliceosomal ATPases/helicases is known. The highly conserved DECD-box protein UAP56/Sub2 is an essential splicing factor that is also important for mRNA export. The expected ATPase/helicase activity appears to be essential for the UAP56/Sub2 functions. Here, we show that purified human UAP56 is an active RNA-dependent ATPase, and we also report the crystal structures ...[more]

PMID: 15585580

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Project description:UL69 of human cytomegalovirus (HCMV) encodes a pleiotropic transactivator protein and has a counterpart in every member of the Herpesviridae family thus far sequenced. However, little is known about the conservation of the functions of the nuclear phosphoprotein pUL69 in the homologous proteins of other betaherpesviruses. Therefore, eukaryotic expression vectors were constructed for pC69 of chimpanzee cytomegalovirus, pRh69 of rhesus cytomegalovirus, pM69 of murine cytomegalovirus, pU42 of human herpesvirus 6, and pU42 of elephant endotheliotropic herpesvirus. Indirect immunofluorescence experiments showed that all pUL69 homologs expressed by these vectors were localized to the cell nucleus. Coimmunoprecipitation experiments identified homodimerization as a conserved feature of all homologs, whereas heterodimerization with pUL69 was restricted to its closer relatives. Further analyses demonstrated that pC69 and pRh69 were the only two homologs that functioned, like pUL69, as viral-mRNA export factors. As we had reported recently that nucleocytoplasmic shuttling and interaction with the cellular DExD/H-box helicases UAP56 and URH49 were prerequisites for the nuclear-mRNA export activity of pUL69, the homologs were characterized with regard to these properties. Heterokaryon assays demonstrated nucleocytoplasmic shuttling for all homologs, and coimmunoprecipitation and mRNA export assays revealed that the interaction of UAP56 and/or URH49 with pC69 or pRh69 was required for mRNA export activity. Moreover, characterization of HCMV recombinants harboring mutations within the N-terminal sequence of pUL69 revealed a strong replication defect of viruses expressing pUL69 variants that were deficient in UAP56 binding. In summary, homodimerization and nucleocytoplasmic shuttling activity were identified as conserved features of betaherpesviral pUL69 homologs. UAP56 binding was shown to represent a unique characteristic of members of the genus Cytomegalovirus that is required for efficient replication of HCMV.

Project description:Human cytomegalovirus (HCMV) encodes the viral mRNA export factor pUL69, which facilitates the cytoplasmic accumulation of mRNA via interaction with the cellular RNA helicase UAP56 or URH49. We reported previously that pUL69 is phosphorylated by cellular CDKs and the viral CDK-like kinase pUL97. Here, we set out to identify phosphorylation sites within pUL69 and to characterize their importance. Mass spectrometry-based phosphosite mapping of pUL69 identified 10 serine/threonine residues as phosphoacceptors. Surprisingly, only a few of these sites localized to the N terminus of pUL69, which could be due to the presence of additional posttranslational modifications, like arginine methylation. As an alternative approach, pUL69 mutants with substitutions of putative phosphosites were analyzed by Phos-tag SDS-PAGE. This demonstrated that serines S46 and S49 serve as targets for phosphorylation by pUL97. Furthermore, we provide evidence that phosphorylation of these serines mediates cis/trans isomerization by the prolyl isomerase Pin1, thus forming a functional Pin1 binding motif. Surprisingly, while abrogation of the Pin1 motif did not affect the replication of recombinant cytomegaloviruses, mutation of serines next to the interaction site for UAP56/URH49 strongly decreased viral replication. This was correlated with a loss of UAP56/URH49 recruitment. Intriguingly, the critical serines S13 and S15 were located within a sequence resembling the UAP56 binding motif (UBM) of cellular mRNA adaptor proteins like REF and UIF. We propose that betaherpesviral mRNA export factors have evolved an extended UAP56/URH49 recognition sequence harboring phosphorylation sites to increase their binding affinities. This may serve as a strategy to successfully compete with cellular mRNA adaptor proteins for binding to UAP56/URH49.IMPORTANCE The multifunctional regulatory protein pUL69 of human cytomegalovirus acts as a viral RNA export factor with a critical role in efficient replication. Here, we identify serine/threonine phosphorylation sites for cellular and viral kinases within pUL69. We demonstrate that the pUL97/CDK phosphosites within alpha-helix 2 of pUL69 are crucial for its cis/trans isomerization by the cellular protein Pin1. Thus, we identified pUL69 as the first HCMV-encoded protein that is phosphorylated by cellular and viral serine/threonine kinases in order to serve as a substrate for Pin1. Furthermore, our study revealed that betaherpesviral mRNA export proteins contain extended binding motifs for the cellular mRNA adaptor proteins UAP56/URH49 harboring phosphorylated serines that are critical for efficient viral replication. Knowledge of the phosphorylation sites of pUL69 and the processes regulated by these posttranslational modifications is important in order to develop antiviral strategies based on a specific interference with pUL69 phosphorylation.

Dataset Information

Crystal structure of the human ATP-dependent splicing and export factor UAP56.

Publications

Crystal structure of the human ATP-dependent splicing and export factor UAP56.

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