scholarly journals pUL69 of Human Cytomegalovirus Recruits the Cellular Protein Arginine Methyltransferase 6 via a Domain That Is Crucial for mRNA Export and Efficient Viral Replication

2015 ◽  
Vol 89 (18) ◽  
pp. 9601-9615 ◽  
Author(s):  
Marco Thomas ◽  
Eric Sonntag ◽  
Regina Müller ◽  
Stefanie Schmidt ◽  
Barbara Zielke ◽  
...  
Keyword(s):  
Secondary Structure ◽  
Human Cytomegalovirus ◽  
Posttranslational Modification ◽  
Mrna Export ◽  
Regulatory Protein ◽  
Arginine Methylation ◽  
Arginine Methyltransferase ◽  
Export Activity ◽  
N Terminus ◽  
Rna Export

ABSTRACTThe regulatory protein pUL69 of human cytomegalovirus acts as a viral mRNA export factor, facilitating the cytoplasmic accumulation of unspliced RNA via interaction with the cellular mRNA export factor UAP56. Here we provide evidence for a posttranslational modification of pUL69 via arginine methylation within the functionally important N terminus. First, we demonstrated a specific immunoprecipitation of full-length pUL69 as well as pUL69aa1-146 by a mono/dimethylarginine-specific antibody. Second, we observed a specific electrophoretic mobility shift upon overexpression of the catalytically active protein arginine methyltransferase 6 (PRMT6). Third, a direct interaction of pUL69 and PRMT6 was confirmed by yeast two-hybrid and coimmunoprecipitation analyses. We mapped the PRMT6 interaction motif to the pUL69 N terminus and identified critical amino acids within the arginine-rich R1 box of pUL69 that were crucial for PRMT6 and/or UAP56 recruitment. In order to test the impact of putative methylation substrates on the functions of pUL69, we constructed various pUL69 derivatives harboring arginine-to-alanine substitutions and tested them for RNA export activity. Thus, we were able to discriminate between arginines within the R1 box of pUL69 that were crucial for UAP56/PRMT6-interaction and/or mRNA export activity. Remarkably, nuclear magnetic resonance (NMR) analyses revealed the same α-helical structures for pUL69 sequences encoding either the wild type R1/R2 boxes or a UAP56/PRMT6 binding-deficient derivative, thereby excluding the possibility that R/A amino acid substitutions within R1 affected the secondary structure of pUL69. We therefore conclude that the pUL69 N terminus is methylated by PRMT6 and that this critically affects the functions of pUL69 for efficient mRNA export and replication of human cytomegalovirus.IMPORTANCEThe UL69 protein of human cytomegalovirus is a multifunctional regulatory protein that acts as a viral RNA export factor with a critical role for efficient replication. Here, we demonstrate that pUL69 is posttranslationally modified via arginine methylation and that the protein methyltransferase PRMT6 mediates this modification. Furthermore, arginine residues with a crucial function for RNA export and for binding of the cellular RNA export factor UAP56 as well as PRMT6 were mapped within the arginine-rich R1 motif of pUL69. Importantly, we demonstrated that mutation of those arginines did not alter the secondary structure of R1, suggesting that they may serve as critical methylation substrates. In summary, our study reveals a novel posttranslational modification of pUL69 which has a significant impact on the function of this important viral regulatory protein. Since PRMTs appear to be amenable to selective inhibition by small molecules, this may constitute a novel target for antiviral therapy.

scholarly journals TAP (NXF1) Belongs to a Multigene Family of Putative RNA Export Factors with a Conserved Modular Architecture

2000 ◽  
Vol 20 (23) ◽  
pp. 8996-9008 ◽  
Author(s):  
Andrea Herold ◽  
Mikita Suyama ◽  
João P. Rodrigues ◽  
Isabelle C. Braun ◽  
Ulrike Kutay ◽  
...  
Keyword(s):  
Drosophila Melanogaster ◽  
Rna Binding ◽  
Mrna Export ◽  
Modular Architecture ◽  
Export Activity ◽  
Evolutionarily Conserved ◽  
Rna Export ◽  
Higher Eukaryotes ◽  
Leucine Rich Repeats ◽  
Rna Export Factors

ABSTRACT 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.

scholarly journals Phosphosite Analysis of the Cytomegaloviral mRNA Export Factor pUL69 Reveals Serines with Critical Importance for Recruitment of Cellular Proteins Pin1 and UAP56/URH49

2020 ◽  
Vol 94 (8) ◽  
Author(s):  
Marco Thomas ◽  
Regina Müller ◽  
Georg Horn ◽  
Boris Bogdanow ◽  
Koshi Imami ◽  
...  
Keyword(s):  
Viral Replication ◽  
Human Cytomegalovirus ◽  
Posttranslational Modifications ◽  
Mrna Export ◽  
Critical Role ◽  
Alpha Helix ◽  
Adaptor Proteins ◽  
Arginine Methylation ◽  
Binding Motif ◽  
Phosphorylation Sites

ABSTRACT 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.

scholarly journals Arginine methyltransferase PRMT5 negatively regulates cGAS-mediated antiviral immune response

2021 ◽  
Vol 7 (13) ◽  
pp. eabc1834
Author(s):  
Dapeng Ma ◽  
Min Yang ◽  
Qiushi Wang ◽  
Caiyu Sun ◽  
Hongbiao Shi ◽  
...  
Keyword(s):  
Immune Response ◽  
Posttranslational Modification ◽  
Arginine Methylation ◽  
Regulatory Effect ◽  
Binding Ability ◽  
Arginine Methyltransferase ◽  
Antiviral Immune Response ◽  
Binding Partner ◽  
Double Stranded Dna ◽  
Direct Binding

Cyclic GMP-AMP synthase (cGAS) functions as an essential DNA sensor, which senses the cytoplasmic double-stranded DNA and activates the antiviral response. However, the posttranslational modification of cGAS remains to be fully understood and whether it has arginine methylation modification remains unknown. Here, we identified protein arginine methyltransferase 5 (PRMT5) as a direct binding partner of cGAS, and it catalyzed the arginine symmetrical dimethylation of cGAS at the Arg124 residue. Further investigation demonstrated that methylation of cGAS by PRMT5 attenuated cGAS-mediated antiviral immune response by blocking the DNA binding ability of cGAS. Oral administration of PRMT5 inhibitors significantly protected mice from HSV-1 infection and prolonged the survival time of these infected mice. Therefore, our findings revealed an essential regulatory effect of PRMT5 on cGAS-mediated antiviral immune response and provided a promising potential antiviral strategy by modulating PRMT5.

scholarly journals Cytomegaloviral protein kinase pUL97 interacts with the nuclear mRNA export factor pUL69 to modulate its intranuclear localization and activity

2009 ◽  
Vol 90 (3) ◽  
pp. 567-578 ◽  
Author(s):  
Marco Thomas ◽  
Sabine Rechter ◽  
Jens Milbradt ◽  
Sabrina Auerochs ◽  
Regina Müller ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Human Cytomegalovirus ◽  
Kinase Inhibitors ◽  
Kinase Inhibitor ◽  
Mrna Export ◽  
Deletion Mapping ◽  
Nuclear Rna ◽  
Rna Export ◽  
Nuclear Rna Export

Human cytomegalovirus encodes a number of phosphorylation-regulated proteins, including the autophosphorylating protein kinase pUL97 and the nuclear mRNA export factor pUL69. Recently, it was reported that the kinase inhibitor roscovitine induces an intranuclear aggregation of pUL69 in infected fibroblasts. Here, we demonstrate that pUL97-specific kinase inhibitors induce a similar pUL69 aggregation. Furthermore, a direct pUL69–pUL97 interaction was demonstrated by coimmunoprecipitation analyses. Deletion mapping identified the domains required for interaction in both proteins (1–140/478–532 in pUL69 and 231–336 in pUL97). Further analysis of the immunoprecipitates by in vitro kinase assays demonstrated the phosphorylation of pUL69 by pUL97. However, catalytically inactive mutants of pUL97 and interaction-negative fragments of pUL69 were phosphorylation-negative. Moreover, an analysis of the pUL69-mediated nuclear RNA export indicated a correlation of the export efficiency with the presence of active pUL97 kinase. These data suggest a specific pUL69–pUL97 interaction and pUL97-mediated phosphorylation which influences the regulatory activities of pUL69.

scholarly journals The UL69 Transactivator Protein of Human Cytomegalovirus Interacts with DEXD/H-Box RNA Helicase UAP56 To Promote Cytoplasmic Accumulation of Unspliced RNA

2006 ◽  
Vol 26 (5) ◽  
pp. 1631-1643 ◽  
Author(s):  
Peter Lischka ◽  
Zsolt Toth ◽  
Marco Thomas ◽  
Regina Mueller ◽  
Thomas Stamminger
Keyword(s):  
Human Cytomegalovirus ◽  
Nuclear Export ◽  
Mrna Export ◽  
Regulatory Protein ◽  
Nuclear Export Signal ◽  
Cellular Transport ◽  
Nucleocytoplasmic Shuttling ◽  
Rna Helicases ◽  
Cytoplasmic Accumulation ◽  
Acid Domain

ABSTRACT The UL69 gene product of human cytomegalovirus belongs to a family of regulatory proteins conserved among all herpesviruses that have in part been characterized as posttranscriptional transactivators participating in the nuclear export of RNA. Recent experiments suggested that pUL69 also acts as a posttranscriptional activator since it was demonstrated that nucleocytoplasmic shuttling via a CRM1-independent nuclear export signal is a prerequisite for its stimulatory effect on gene expression. Based on these findings we initiated studies to investigate the role of pUL69 in mRNA export and demonstrate that pUL69 efficiently promotes the cytoplasmic accumulation of unspliced RNA. Furthermore, we show that this pUL69 activity is linked to the cellular mRNA export machinery by direct protein interaction with the highly related DEXD/H-box RNA helicases UAP56 and URH49. Particularly, we identified a 12-amino-acid domain within the N terminus of pUL69 which is required for binding to UAP56 and URH49, and we could demonstrate that UAP56 interaction and nucleocytoplasmic shuttling are both prerequisites for pUL69-mediated mRNA export. Thus, we identified a novel cellular target which provides a herpesviral regulatory protein with access to a conserved cellular transport system in order to promote nuclear export of unspliced RNA.

scholarly journals PTEN arginine methylation by PRMT6 suppresses PI3K–AKT signaling and modulates pre-mRNA splicing

2019 ◽  
Vol 116 (14) ◽  
pp. 6868-6877 ◽  
Author(s):  
Jiawen Feng ◽  
Yaping Dang ◽  
Weiqi Zhang ◽  
Xuyang Zhao ◽  
Cong Zhang ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Alternative Splicing ◽  
Posttranslational Modification ◽  
Arginine Methylation ◽  
Akt Signaling ◽  
Mrna Splicing ◽  
Mass Spectrometry Analysis ◽  
Arginine Methyltransferase ◽  
Spectrometry Analysis ◽  
Cellular Processes

Arginine methylation is a ubiquitous posttranslational modification that regulates critical cellular processes including signal transduction and pre-mRNA splicing. Here, we report that the tumor-suppressor PTEN is methylated by protein arginine methyltransferase 6 (PRMT6). Mass-spectrometry analysis reveals that PTEN is dimethylated at arginine 159 (R159). We found that PTEN is mutated at R159 in cancers, and the PTEN mutant R159K loses its capability to inhibit the PI3K–AKT cascade. Furthermore, PRMT6 is physically associated with PTEN, promotes asymmetrical dimethylation of PTEN, and regulates the PI3K–AKT cascade through PTEN R159 methylation. In addition, using transcriptome analyses, we found that PTEN R159 methylation is involved in modulation of pre-mRNA alternative splicing. Our results demonstrate that PTEN is functionally regulated by arginine methylation. We propose that PTEN arginine methylation modulates pre-mRNA alternative splicing and influences diverse physiologic processes.

scholarly journals Roles for RNA export factor, Nxt1, in ensuring muscle integrity and normal RNA expression in Drosophila

2020 ◽  
Vol 11 (1) ◽  
Author(s):  
Kevin van der Graaf ◽  
Katia Jindrich ◽  
Robert Mitchell ◽  
Helen White-Cooper
Keyword(s):  
Mrna Export ◽  
Rna Stability ◽  
Muscle Degeneration ◽  
Loss Of Function ◽  
Cellular Functions ◽  
Partial Loss ◽  
Export Pathway ◽  
Pathway Gene ◽  
Rna Export

Abstract The mRNA export pathway is responsible for the transport of mRNAs from the nucleus to the cytoplasm, and thus is essential for protein production and normal cellular functions. A partial loss of function allele of the mRNA export factor Nxt1 in Drosophila shows reduced viability and sterility. A previous study has shown that the male fertility defect is due to a defect in transcription and RNA stability, indicating the potential for this pathway to be implicated in processes beyond the known mRNA transport function. Here we investigate the reduced viability of Nxt1 partial loss of function mutants, and describe a defect in growth and maintenance of the larval muscles, leading to muscle degeneration. RNA-seq revealed reduced expression of a set of mRNAs, particularly from genes with long introns in Nxt1 mutant carcass. We detected differential expression of circRNA, and significantly fewer distinct circRNAs expressed in the mutants. Despite the widespread defects in gene expression, muscle degeneration was rescued by increased expression of the costamere component tn (abba) in muscles. This is the first report of a role for the RNA export pathway gene Nxt1 in the maintenance of muscle integrity. Our data also links the mRNA export pathway to a specific role in the expression of mRNA and circRNA from common precursor genes, in vivo.

scholarly journals The Role of Protein Arginine Methyltransferase 1 in Gastrointestinal Cancers

2021 ◽  
Author(s):  
Jin Zou ◽  
Wei Shen ◽  
Yu Zhang ◽  
Shibo Ying
Keyword(s):  
Tumor Cells ◽  
Cancer Progression ◽  
Cancer Metastasis ◽  
Arginine Methylation ◽  
Gastrointestinal Tumors ◽  
Migration And Invasion ◽  
Arginine Methyltransferase ◽  
Tumorigenic Mechanism ◽  
Methyltransferase 1

Mammals can produce nine kinds of arginine methylation enzymes that can be divided into three types (I, II, and III) according to their catalytic activity. Arginine methyltransferase 1 (PRMT1), as the first discovered arginine methyltransferase type I, has been reported to be involved in cell signal transduction, DNA damage repair, RNA transcription and other processes. Its imbalance or abnormal expression is also involved in cancer metastasis. PRMT1 is highly expressed in gastrointestinal tumors and promotes tumor biomarkers expression, chemotherapy resistance and tumorigenicity to promote cancer progression, while downregulation of PRMT1 expression can inhibit the migration and invasion of related tumor cells or promote tumor cells apoptosis and inhibit the progression of cancer. Therefore, PRMT1 may be a cancer therapeutic target. In this paper, arginine methylase 1 expression in various types of gastrointestinal tumors, the tumorigenic mechanism and the role of PRMT1 in tumorigenesis and development were reviewed.

scholarly journals Modification of the 5′ Terminus of Sindbis Virus Genomic RNA Allows nsP4 RNA Polymerases with Nonaromatic Amino Acids at the N Terminus To Function in RNA Replication

2003 ◽  
Vol 77 (4) ◽  
pp. 2301-2309 ◽  
Author(s):  
Yukio Shirako ◽  
Ellen G. Strauss ◽  
James H. Strauss
Keyword(s):  
Amino Acid ◽  
Secondary Structure ◽  
Sindbis Virus ◽  
Rna Synthesis ◽  
Wild Type Virus ◽  
Progeny Virus ◽  
Minus Strand ◽  
Genomic Rna ◽  
Wild Type ◽  
N Terminus

ABSTRACT We have previously shown that Sindbis virus RNA polymerase requires an N-terminal aromatic amino acid or histidine for wild-type or pseudo-wild-type function; mutant viruses with a nonaromatic amino acid at the N terminus of the polymerase, but which are otherwise wild type, are unable to produce progeny viruses and will not form a plaque at any temperature tested. We now show that such mutant polymerases can function to produce progeny virus sufficient to form plaques at both 30 and 40°C upon addition of AU, AUA, or AUU to the 5′ terminus of the genomic RNA or upon substitution of A for U as the third nucleotide of the genome. These results are consistent with the hypothesis that (i) 3′-UA-5′ is required at the 3′ terminus of the minus-strand RNA for initiation of plus-strand genomic RNA synthesis; (ii) in the wild-type virus this sequence is present in a secondary structure that can be opened by the wild-type polymerase but not by the mutant polymerase; (iii) the addition of AU, AUA, or AUU to the 5′ end of the genomic RNA provides unpaired 3′-UA-5′ at the 3′ end of the minus strand that can be utilized by the mutant polymerase, and similarly, the effect of the U3A mutation is to destabilize the secondary structure, freeing 3′-terminal UA; and (iv) the N terminus of nsP4 may directly interact with the 3′ terminus of the minus-strand RNA for the initiation of the plus-strand genomic RNA synthesis. This hypothesis is discussed in light of our present results as well as of previous studies of alphavirus RNAs, including defective interfering RNAs.

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