scholarly journals Multimerization of human cytomegalovirus regulatory protein UL69 via a domain that is conserved within its herpesvirus homologues

2007 ◽  
Vol 88 (2) ◽  
pp. 405-410 ◽  
Author(s):  
Peter Lischka ◽  
Marco Thomas ◽  
Zsolt Toth ◽  
Regina Mueller ◽  
Thomas Stamminger
Keyword(s):  
Human Cytomegalovirus ◽  
Nuclear Export ◽  
Rna Binding ◽  
Regulatory Protein ◽  
Transcriptional Level ◽  
Homologous Proteins ◽  
Interaction Domain ◽  
Self Association

The UL69 protein of human cytomegalovirus is a multifunctional regulatory protein that has counterparts in all herpesviruses. Some of these proteins have been shown to function primarily at the post-transcriptional level in promoting nuclear export of viral transcripts. Consistently, this group has reported recently that pUL69 is an RNA-binding, nucleocytoplasmic shuttling protein that facilitates the cytoplasmic accumulation of unspliced mRNA via its interaction with the cellular mRNA export factor UAP56. Evidence has been presented to suggest that some of the pUL69 homologues self-interact and function in vivo as multimers. Herein, the possibility of pUL69 self-association was examined and it has been demonstrated that pUL69 can interact with itself in vitro and in vivo in order to form high-molecular-mass complexes. The self-interaction domain within pUL69 was mapped to a central domain of this viral protein that is conserved within the homologous proteins of other herpesviruses, suggesting that multimerization is a conserved feature of this protein family.

scholarly journals Functional Interaction between Pleiotropic Transactivator pUL69 of Human Cytomegalovirus and the Human Homolog of Yeast Chromatin Regulatory Protein SPT6

2000 ◽  
Vol 74 (17) ◽  
pp. 8053-8064 ◽  
Author(s):  
Michael Winkler ◽  
Thomas aus dem Siepen ◽  
Thomas Stamminger
Keyword(s):  
Human Cytomegalovirus ◽  
Viral Protein ◽  
Regulatory Protein ◽  
Single Amino Acid ◽  
Human Homolog ◽  
Carboxy Terminus ◽  
Homologous Proteins ◽  
Interaction Domain ◽  
Central Domain

ABSTRACT The phosphoprotein pUL69 of human cytomegalovirus (HCMV), which is a herpesvirus of considerable medical importance in immunosuppressed patients and newborns, has previously been identified as an early-late viral protein that can stimulate several viral and cellular promoters and thus exerts a rather broad activation pattern. To gain insight into the mechanism of this transactivation process, we looked for cellular factors interacting with pUL69 in a yeast two-hybrid screen. Using a B-lymphocyte cDNA library fused to the GAL4 activation domain, we identified 34 clones, 11 of which comprised one distinct gene. Interaction with this gene turned out to be very strong, producing β-galactosidase levels 100-fold greater than the background as measured in an ONPG (o-nitrophenyl-β-d-galactopyranoside) assay. Sequencing identified this gene as the human homolog of the yeast factor SPT6, which is thought to be involved in the regulation of chromatin structure. A direct interaction of pUL69 and the carboxy terminus of hSPT6 could be demonstrated using in vitro pull-down experiments. After having generated a specific antiserum that is able to detect the endogenous hSPT6 protein, we were able to observe an in vivo interaction of both proteins by coimmunoprecipitation analysis. The interaction domain within pUL69 was mapped to a central domain of this viral protein that is conserved within the homologous proteins of other herpesviruses such as the ICP27 protein of herpes simplex virus. Internal deletions within this central domain, as well as a single amino acid exchange at position C495, resulted in a loss of interaction. This correlated with a loss of the transactivation potential of the respective mutants, suggesting that the hSPT6 interaction of pUL69 is essential for stimulating gene expression. Furthermore, we demonstrate that the carboxy terminus of hSPT6 also binds to histon H3 and that this interaction can be antagonized by pUL69. This allows the deduction of a model by which pUL69 acts as an antirepressor by competing for binding of histones to hSPT6, thereby antagonizing the chromatin remodeling function of this cellular protein.

scholarly journals General and Target-Specific RNA Binding Properties of Epstein-Barr Virus SM Posttranscriptional Regulatory Protein

2009 ◽  
Vol 83 (22) ◽  
pp. 11635-11644 ◽  
Author(s):  
Zhao Han ◽  
Dinesh Verma ◽  
Chelsey Hilscher ◽  
Dirk P. Dittmer ◽  
Sankar Swaminathan
Keyword(s):  
Gene Expression ◽  
Nuclear Export ◽  
Epstein Barr Virus ◽  
Rna Binding ◽  
Regulatory Protein ◽  
Lytic Cycle ◽  
Binding Properties ◽  
Barr Virus ◽  
Epstein Barr

ABSTRACT Epstein-Barr virus (EBV) SM protein is an essential nuclear shuttling protein expressed by EBV early during the lytic phase of replication. SM acts to increase EBV lytic gene expression by binding EBV mRNAs and enhancing accumulation of the majority of EBV lytic cycle mRNAs. SM increases target mRNA stability and nuclear export, in addition to modulating RNA splicing. SM and its homologs in other herpesvirus have been hypothesized to function in part by binding viral RNAs and recruiting cellular export factors. Although activation of gene expression by SM is gene specific, it is unknown whether SM binds to mRNA in a specific manner or whether its RNA binding is target independent. SM-mRNA complexes were isolated from EBV-infected B-lymphocyte cell lines induced to permit lytic EBV replication, and a quantitative measurement of mRNAs corresponding to all known EBV open reading frames was performed by real-time quantitative reverse transcription-PCR. The results showed that although SM has broad RNA binding properties, there is a clear hierarchy of affinities among EBV mRNAs with respect to SM complex formation. In vitro binding assays with two of the most highly SM-associated transcripts suggested that SM binds preferentially to specific sequences or structures present in noncoding regions of some EBV mRNAs. Furthermore, the presence of these sequences conferred responsiveness to SM. These data are consistent with a mechanism of action similar to that of hnRNPs, which exert sequence-specific effects on gene expression despite having multiple degenerate consensus binding sites common to a large number of RNAs.

scholarly journals Transcriptional Pausing in the Bacillus subtilis pyr Operon In Vitro: a Role in Transcriptional Attenuation?

2003 ◽  
Vol 185 (16) ◽  
pp. 4764-4771 ◽  
Author(s):  
Hesheng Zhang ◽  
Robert L. Switzer
Keyword(s):  
Bacillus Subtilis ◽  
Rna Binding ◽  
Bacterial Species ◽  
Regulatory Protein ◽  
Stem Loop ◽  
Nucleotide Biosynthesis ◽  
Pyrimidine Nucleotide ◽  
Transcriptional Pausing

ABSTRACT The genes encoding the enzymes of pyrimidine nucleotide biosynthesis (pyr genes) are regulated in Bacillus subtilis and many other bacterial species by transcriptional attenuation. When UMP or UTP is bound to the PyrR regulatory protein, it binds to pyr mRNA at specific sequences and secondary structures in the RNA. Binding to this site prevents formation of an antiterminator stem-loop in the RNA and permits formation of a downstream terminator, leading to reduced expression of the pyr genes lying downstream from the terminator. The functioning of several other transcriptional attenuation systems has been shown to involve transcriptional pausing; this observation stimulated us to use single-round transcription of pyr genes to test for formation of paused transcripts in vitro. Using templates with each of the three known B. subtilis pyr attenuation sites, we identified one major pause site in each in which the half-life of the paused transcript was increased four- to sixfold by NusA. In each case pausing at the NusA-stimulated site prevented formation of a complete antiterminator stem-loop, while it resulted in increased time for a PyrR binding loop to form and for PyrR to bind to this loop. Thus, the pausing detected in vitro is potentially capable of playing a role in establishing the correct timing for pyr attenuation in vivo. With two of three pyr templates the combination of NusA with PyrR markedly stimulated termination of transcription at the normal termination sites. This suggests that NusA, by stabilizing pausing, plays a role in termination of pyr transcription in vivo.

scholarly journals NCBP3 positively impacts mRNA biogenesis

2020 ◽  
Vol 48 (18) ◽  
pp. 10413-10427 ◽  
Author(s):  
Yuhui Dou ◽  
Isabelle Barbosa ◽  
Hua Jiang ◽  
Claudia Iasillo ◽  
Kelly R Molloy ◽  
...  
Keyword(s):  
Rna Polymerase Ii ◽  
Nuclear Export ◽  
Rna Binding ◽  
Rna Degradation ◽  
Exon Junction Complex ◽  
Protein Protein Interaction ◽  
Cap Binding Complex ◽  
Interaction Screening

Abstract The nuclear Cap-Binding Complex (CBC), consisting of Nuclear Cap-Binding Protein 1 (NCBP1) and 2 (NCBP2), associates with the nascent 5′cap of RNA polymerase II transcripts and impacts RNA fate decisions. Recently, the C17orf85 protein, also called NCBP3, was suggested to form an alternative CBC by replacing NCBP2. However, applying protein–protein interaction screening of NCBP1, 2 and 3, we find that the interaction profile of NCBP3 is distinct. Whereas NCBP1 and 2 identify known CBC interactors, NCBP3 primarily interacts with components of the Exon Junction Complex (EJC) and the TRanscription and EXport (TREX) complex. NCBP3-EJC association in vitro and in vivo requires EJC core integrity and the in vivo RNA binding profiles of EJC and NCBP3 overlap. We further show that NCBP3 competes with the RNA degradation factor ZC3H18 for binding CBC-bound transcripts, and that NCBP3 positively impacts the nuclear export of polyadenylated RNAs and the expression of large multi-exonic transcripts. Collectively, our results place NCBP3 with the EJC and TREX complexes in supporting mRNA expression.

scholarly journals Formation of a Tap/NXF1 Homotypic Complex Is Mediated through the Amino-Terminal Domain of Tap and Enhances Interaction with Nucleoporins

2008 ◽  
Vol 19 (1) ◽  
pp. 327-338 ◽  
Author(s):  
Leah H. Matzat ◽  
Stephen Berberoglu ◽  
Lyne Lévesque
Keyword(s):  
Nuclear Export ◽  
Rna Binding ◽  
Direct Interaction ◽  
Nuclear Pore ◽  
Amino Terminal ◽  
Multimeric Complex ◽  
Rna Export ◽  
Amino Terminal Domain

Nuclear export of mRNAs is mediated by the Tap/Nxt1 pathway. Tap moves its RNA cargo through the nuclear pore complex by direct interaction with nucleoporin phenylalanine-glycine repeats. This interaction is strengthened by the formation of a Tap/Nxt1 heterodimer. We now present evidence that Tap can form a multimeric complex with itself and with other members of the NXF family. We also show that the homotypic Tap complex can interact with both Nxt1 and nucleoporins in vitro. The region mediating this oligomerization is localized to the first 187 amino acids of Tap, which overlaps with its RNA-binding domain. Removal of this domain greatly reduces the ability of Tap to bind nucleoporins in vitro and in vivo. This is the first report showing that the Tap amino terminus modulates the interaction of Tap with nucleoporins. We speculate that this mechanism has a regulatory role for RNA export independent of RNA binding.

scholarly journals Efficient Nuclear Export of Herpes Simplex Virus 1 Transcripts Requires both RNA Binding by ICP27 and ICP27 Interaction with TAP/NXF1

2008 ◽  
Vol 83 (3) ◽  
pp. 1184-1192 ◽  
Author(s):  
Lisa A. Johnson ◽  
Rozanne M. Sandri-Goldin
Keyword(s):  
Herpes Simplex Virus ◽  
Herpes Simplex ◽  
Nuclear Export ◽  
Rna Binding ◽  
Regulatory Protein ◽  
Herpes Simplex Virus 1 ◽  
Cytoplasmic Rna ◽  
Simplex Virus ◽  
Hsv 1

ABSTRACT Herpes simplex virus 1 (HSV-1) regulatory protein ICP27 has been reported to bind viral RNA and to interact with the nuclear export adaptor Aly/REF and the major cellular mRNA nuclear export receptor TAP/NXF1. Using in situ hybridization and in vitro export assays, we show here that poly(A)+ RNA was retained in the nucleus of cells infected with viral ICP27 mutants that either cannot bind RNA or that do not interact with TAP/NXF1. Microarray analysis of nuclear and cytoplasmic RNA fractions demonstrated that efficient export of the majority of viral transcripts requires that ICP27 be able to bind RNA and to interact with TAP/NXF1. We conclude that ICP27 is the major export adaptor for HSV-1 mRNA and that it links bound transcripts to the TAP/NXF1 export receptor.

scholarly journals The Mechanism of Nuclear Export of Smad3 Involves Exportin 4 and Ran

2006 ◽  
Vol 26 (4) ◽  
pp. 1318-1332 ◽  
Author(s):  
Akira Kurisaki ◽  
Keiko Kurisaki ◽  
Marcin Kowanetz ◽  
Hiromu Sugino ◽  
Yoshihiro Yoneda ◽  
...  
Keyword(s):  
Transforming Growth Factor Beta ◽  
Nuclear Export ◽  
Transforming Growth Factor ◽  
Peptide Sequence ◽  
Nuclear Accumulation ◽  
Dependent Manner ◽  
Nucleotide Exchange ◽  
Interaction Domain

ABSTRACT Transforming growth factor beta (TGF-β) receptors phosphorylate Smad3 and induce its nuclear import so it can regulate gene transcription. Smad3 can return to the cytoplasm to propagate further cycles of signal transduction or to be degraded. We demonstrate that Smad3 is exported by a constitutive mechanism that is insensitive to leptomycin B. The Mad homology 2 (MH2) domain is responsible for Smad3 export, which requires the GTPase Ran. Inactive, GDP-locked RanT24N or nuclear microinjection of Ran GTPase activating protein 1 blocked Smad3 export. Inactivation of the Ran guanine nucleotide exchange factor RCC1 inhibited Smad3 export and led to nuclear accumulation of phosphorylated Smad3. A screen for importin/exportin family members that associate with Smad3 identified exportin 4, which binds a conserved peptide sequence in the MH2 domain of Smad3 in a Ran-dependent manner. Exportin 4 is sufficient for carrying the in vitro nuclear export of Smad3 in cooperation with Ran. Knockdown of endogenous exportin 4 completely abrogates the export of endogenous Smad3. A short peptide representing the minimal interaction domain in Smad3 effectively competes with Smad3 association to exportin 4 and blocks nuclear export of Smad3 in vivo. We thus delineate a novel nuclear export pathway for Smad3.

scholarly journals Self-association of the single-KH-domain family members Sam68, GRP33, GLD-1, and Qk1: role of the KH domain.

1997 ◽  
Vol 17 (10) ◽  
pp. 5707-5718 ◽  
Author(s):  
T Chen ◽  
B B Damaj ◽  
C Herrera ◽  
P Lasko ◽  
S Richard
Keyword(s):  
Mammalian Cells ◽  
Rna Binding ◽  
Family Members ◽  
Artemia Salina ◽  
Domain Family ◽  
Protein Motifs ◽  
Kh Domain ◽  
Self Association

Sam68 is a member of a growing family of proteins that contain a single KH domain embedded in a larger conserved domain of approximately 170 amino acids. Loops 1 and 4 of this KH domain family are longer than the corresponding loops in other KH domains and contain conserved residues. KH domains are protein motifs that are involved in RNA binding and are often present in multiple copies. Here we demonstrate by coimmunoprecipitation studies that Sam68 self-associated and that cellular RNA was required for the association. Deletion studies demonstrated that the Sam68 KH domain loops 1 and 4 were required for self-association. The Sam68 interaction was also observed in Saccharomyces cerevisiae by the two-hybrid system. In situ chemical cross-linking studies in mammalian cells demonstrated that Sam68 oligomerized in vivo. These Sam68 complexes bound homopolymeric RNA and the SH3 domains of p59fyn and phospholipase Cgamma1 in vitro, demonstrating that Sam68 associates with RNA and signaling molecules as a multimer. The formation of the Sam68 complex was inhibited by p59fyn, suggesting that tyrosine phosphorylation regulates Sam68 oligomerization. Other Sam68 family members including Artemia salina GRP33, Caenorhabditis elegans GLD-1, and mouse Qk1 also oligomerized. In addition, Sam68, GRP33, GLD-1, and Qk1 associated with other KH domain proteins such as Bicaudal C. These observations indicate that the single KH domain found in the Sam68 family, in addition to mediating protein-RNA interactions, mediates protein-protein interactions.

scholarly journals In vitro reconstitution of an mRNA-transport complex reveals mechanisms of assembly and motor activation

2013 ◽  
Vol 203 (6) ◽  
pp. 971-984 ◽  
Author(s):  
Roland G. Heym ◽  
Dennis Zimmermann ◽  
Franziska T. Edelmann ◽  
Lars Israel ◽  
Zeynep Ökten ◽  
...  
Keyword(s):  
Nuclear Export ◽  
Messenger Rna ◽  
Rna Binding ◽  
Long Distance ◽  
Long Distance Transport ◽  
In Vitro Reconstitution ◽  
Localization Elements ◽  
Transport Complex

The assembly and composition of ribonucleic acid (RNA)–transporting particles for asymmetric messenger RNA (mRNA) localization is not well understood. During mitosis of budding yeast, the Swi5p-dependent HO expression (SHE) complex transports a set of mRNAs into the daughter cell. We recombinantly reconstituted the core SHE complex and assessed its properties. The cytoplasmic precomplex contains only one motor and is unable to support continuous transport. However, a defined interaction with a second, RNA-bound precomplex after its nuclear export dimerizes the motor and activates processive RNA transport. The run length observed in vitro is compatible with long-distance transport in vivo. Surprisingly, SHE complexes that either contain or lack RNA cargo show similar motility properties, demonstrating that the RNA-binding protein and not its cargo activates motility. We further show that SHE complexes have a defined size but multimerize into variable particles upon binding of RNAs with multiple localization elements. Based on these findings, we provide an estimate of number, size, and composition of such multimeric SHE particles in the cell.

scholarly journals An RNA-binding region regulates CTCF clustering and chromatin looping

2018 ◽  
Author(s):  
Anders S. Hansen ◽  
Tsung-Han S. Hsieh ◽  
Claudia Cattoglio ◽  
Iryna Pustova ◽  
Xavier Darzacq ◽  
...  
Keyword(s):  
Rna Binding ◽  
Embryonic Stem ◽  
Loop Formation ◽  
Cell Type ◽  
Binding Region ◽  
Chromatin Loops ◽  
Cell Type Specific ◽  
Self Association

Mammalian genomes are folded into Topologically Associating Domains (TADs), consisting of cell-type specific chromatin loops anchored by CTCF and cohesin. Since CTCF and cohesin are expressed ubiquitously, how cell-type specific CTCF-mediated loops are formed poses a paradox. Here we show RNase-sensitive CTCF self-association in vitro and that an RNA-binding region (RBR) mediates CTCF clustering in vivo. Intriguingly, deleting the RBR abolishes or impairs almost half of all chromatin loops in mouse embryonic stem cells. Disrupted loop formation correlates with abrogated clustering and diminished chromatin binding of the RBR mutant CTCF protein, which in turn results in a failure to halt cohesin-mediated extrusion. Thus, CTCF loops fall into at least 2 classes: RBR-independent and RBR-dependent loops. We suggest that evidence for distinct classes of RBR-dependent loops may provide a mechanism for establishing cell-specific CTCF loops regulated by RNAs and other RBR partner.

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