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Arginine methyltransferase affects interactions and recruitment of mRNA processing and export factors.

ABSTRACT: Hmt1 is the major type I arginine methyltransferase in the yeast Saccharomyces cerevisiae and facilitates the nucleocytoplasmic transport of mRNA-binding proteins through their methylation. Here we demonstrate that Hmt1 is recruited during the beginning of the transcriptional elongation process. Hmt1 methylates Yra1 and Hrp1, two mRNA-binding proteins important for mRNA processing and export. Moreover, loss of Hmt1 affects interactions between mRNA-binding proteins and Tho2, a component of the TREX (transcription/export) complex that is important for transcriptional elongation and recruitment of mRNA export factors. Furthermore, RNA in situ hybridization analysis demonstrates that loss of Hmt1 results in slowed release of HSP104 mRNA from the sites of transcription. Genome-wide location analysis shows that Hmt1 is bound to specific functional gene classes, many of which are also bound by Tho2 and other mRNA-processing factors. These data suggest a model whereby Hmt1 affects transcriptional elongation and, as a result, influences recruitment of RNA-processing factors.

SUBMITTER: Yu MC

PROVIDER: S-EPMC514182 | biostudies-other | 2004 Aug

REPOSITORIES: biostudies-other

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Project description:UnlabelledThe 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.

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Arginine methyltransferase affects interactions and recruitment of mRNA processing and export factors.

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