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      The Immunosuppressive Agent Mizoribine Monophosphate Is an Inhibitor of the Human RNA Capping Enzyme


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          Mizoribine monophosphate (MZP) is a specific inhibitor of the cellular inosine-5′-monophosphate dehydrogenase (IMPDH), the enzyme catalyzing the rate-limiting step of de novo guanine nucleotide biosynthesis. MZP is a highly potent antagonistic inhibitor of IMPDH that blocks the proliferation of T and B lymphocytes that use the de novo pathway of guanine nucleotide synthesis almost exclusively. In the present study, we investigated the ability of MZP to directly inhibit the human RNA capping enzyme (HCE), a protein harboring both RNA 5′-triphosphatase and RNA guanylyltransferase activities. HCE is involved in the synthesis of the cap structure found at the 5′ end of eukaryotic mRNAs, which is critical for the splicing of the cap-proximal intron, the transport of mRNAs from the nucleus to the cytoplasm, and for both the stability and translation of mRNAs. Our biochemical studies provide the first insight that MZP can inhibit the formation of the RNA cap structure catalyzed by HCE. In the presence of MZP, the RNA 5′-triphosphatase activity appears to be relatively unaffected while the RNA guanylyltransferase activity is inhibited, indicating that the RNA guanylyltransferase activity is the main target of MZP inhibition. Kinetic studies reveal that MZP is a non-competitive inhibitor that likely targets an allosteric site on HCE. Mizoribine also impairs mRNA capping in living cells, which could account for the global mechanism of action of this therapeutic agent. Together, our study clearly demonstrates that mizoribine monophosphate inhibits the human RNA guanylyltransferase in vitro and impair mRNA capping in cellulo.

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          Most cited references36

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          Different phosphorylated forms of RNA polymerase II and associated mRNA processing factors during transcription.

          The activities of several mRNA processing factors are coupled to transcription through binding to RNA polymerase II (Pol II). The largest subunit of Pol II contains a repetitive carboxy-terminal domain (CTD) that becomes highly phosphorylated during transcription. mRNA-capping enzyme binds only to phosphorylated CTD, whereas other processing factors may bind to both phosphorylated and unphosphorylated forms. Capping occurs soon after transcription initiation and before other processing events, raising the question of whether capping components remain associated with the transcription complex after they have modified the 5' end of the mRNA. Chromatin immunoprecipitation in Saccharomyces cerevisiae shows that capping enzyme cross-links to promoters but not coding regions. In contrast, the mRNA cap methyltransferase and the Hrp1/CFIB polyadenylation factor cross-link to both promoter and coding regions. Remarkably, the phosphorylation pattern of the CTD changes during transcription. Ser 5 phosphorylation is detected primarily at promoter regions dependent on TFIIH. In contrast, Ser 2 phosphorylation is seen only in coding regions. These results suggest a dynamic association of mRNA processing factors with differently modified forms of the polymerase throughout the transcription cycle.
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            The enzymes and control of eukaryotic mRNA turnover.

            The degradation of eukaryotic mRNAs plays important roles in the modulation of gene expression, quality control of mRNA biogenesis and antiviral defenses. In the past five years, many of the enzymes involved in this process have been identified and mechanisms that modulate their activities have begun to be identified. In this review, we describe the enzymes of mRNA degradation and their properties. We highlight that there are a variety of enzymes with different specificities, suggesting that individual nucleases act on distinct subpopulations of transcripts within the cell. In several cases, translation factors that bind mRNA inhibit these nucleases. In addition, recent work has begun to identify distinct mRNP complexes that recruit the nucleases to transcripts through different mRNA-interacting proteins. These properties and complexes suggest multiple mechanisms by which mRNA degradation could be regulated.
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              Distinction and relationship between elongation rate and processivity of RNA polymerase II in vivo.

              A number of proteins and drugs have been implicated in the process of transcriptional elongation by RNA polymerase (Pol) II, but the factors that govern the elongation rate (nucleotide additions per min) and processivity (nucleotide additions per initiation event) in vivo are poorly understood. Here, we show that a mutation in the Rpb2 subunit of Pol II reduces both the elongation rate and processivity in vivo. In contrast, none of the putative elongation factors tested affect the elongation rate, although mutations in the THO complex and in Spt4 significantly reduce processivity. The drugs 6-azauracil and mycophenolic acid reduce both the elongation rate and processivity, and this processivity defect is aggravated by mutations in Spt4, TFIIS, and CTDK-1. Our results suggest that, in vivo, a reduced rate of Pol II elongation leads to premature dissociation along the chromatin template and that Pol II processivity can be uncoupled from elongation rate.

                Author and article information

                Role: Editor
                PLoS One
                PLoS ONE
                PLoS ONE
                Public Library of Science (San Francisco, USA )
                17 January 2013
                : 8
                : 1
                : e54621
                [1 ]Département de Biochimie, Faculté de Médecine et des Sciences de la Santé, Université de Sherbrooke, Sherbrooke, Québec, Canada
                [2 ]Graduate School of Pharmaceutical Sciences, Hokkaido University, Sapporo, Japan
                Kantonal Hospital St. Gallen, Switzerland
                Author notes

                Competing Interests: The authors have declared that no competing interests exist.

                Conceived and designed the experiments: FPJ MB. Performed the experiments: FPJ IB. Analyzed the data: FPJ MB. Contributed reagents/materials/analysis tools: SS. Wrote the paper: FPJ MB.


                This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

                : 7 August 2012
                : 13 December 2012
                Page count
                Pages: 11
                This work was supported by grants from the Canadian Institutes of Health Research ( http://www.cihr-irsc.gc.ca) and the Natural Sciences and Engineering Research Council ( http://www.nserc-crsng.gc.ca). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
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