Synthesis, Conformation and Hydrolytic Stability of p<sup>1</sup>,p<sup>3</sup>−Dinucleoside Triphosphates Related to mRNA 5′-cap, and Comparative Kinetic Studies on their Nucleoside and Nucleoside Monophosphate Analogs

Darżynkiewicz, Edward; Stępiński, Janusz; Tahara, Stanley M.; Stolarski, Ryszard; Ekiel, Irena; Haber, Dorota; Neuvonen, Kari; Lehikoinen, Pertti; Labádi, I.; Lönnberg, Harri

doi:10.1080/07328319008045191

Cited by 50 publications

(45 citation statements)

References 28 publications

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“…Cap Analogs-Synthesis of the cap analogs Gp 3 G, m 7 Gp 3 G, b 7 Gp 3 G, and m 2 7,3Ј-O Gp 3 G has been described previously (53)(54)(55)(56). Two new cap analogs were designed and synthesized for this study, m 2 7,3Ј-O Gp CH2 ppG and m 2 7,3Ј-O Gpp CH2 pG.…”

Section: Methodsmentioning

confidence: 99%

Differential Inhibition of mRNA Degradation Pathways by Novel Cap Analogs

Grudzien

Kalek

Jemielity

et al. 2006

Journal of Biological Chemistry

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113

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mRNA degradation predominantly proceeds through two alternative routes: the 533 pathway, which requires deadenylation followed by decapping and 533 hydrolysis; and the 335 pathway, which involves deadenylation followed by 335 hydrolysis and finally decapping. The mechanisms and relative contributions of each pathway are not fully understood. We investigated the effects of different cap structure (Gp 3 G, m 7 Gp 3 G, or m 2 7,3-O Gp 3 G) and 3 termini (A 31 , A 60 , or G 16 ) on both translation and mRNA degradation in mammalian cells. The results indicated that cap structures that bind eIF4E with higher affinity stabilize mRNA to degradation in vivo. mRNA stability depends on the ability of the 5 terminus to bind eIF4E, not merely the presence of a blocking group at the 5-end. Introducing a stem-loop in the 5-UTR that dramatically reduces translation, but keeping the cap structure the same, does not alter the rate of mRNA degradation. The 5Ј terminus of all eukaryotic cellular mRNAs is modified with a 5Ј-5Ј m 7 GTP-containing cap (1). Caps fulfill a variety of functions in the synthesis, translation, and degradation of mRNA. The presence of the 5Ј cap structure increases both the accuracy and efficiency of pre-mRNA splicing (2, 3). The cap on pre-mRNA interacts with the nuclear cap-binding complex, which remains bound and plays an active role during RNA processing and export (4). In the cytosol, the cap structure is required for efficient translation of mRNA. The cap is specifically recognized by the translational initiation factor eIF4E (5, 6). Binding of eIF4E to the cap occurs during formation of the 48 S initiation complex, which is rate limiting for translation initiation under normal conditions (7,8). Finally, the cap serves as one determinant of mRNA degradation. Capped mRNAs are more stable than their uncapped counterparts (9). The cap structure helps to protect RNA from degradation by 5Ј33Ј-exonucleases located in the cytosol and nucleus, as demonstrated in both Saccharomyces cerevisiae (10) and mammalian cells (11,12).A second stability element in mRNA is the 3Ј-terminal poly(A) tract. PABP 2,3 binds to poly(A) and is essential for the stability provided by this element, protecting mRNA against exonucleolytic degradation (12-15). PABP also binds to the N terminus of eIF4G (16) and stabilizes the eIF4G⅐eIF4E complex, enhancing translational reinitiation (17, 18). The stimulation conferred by the cap and poly(A) tract are synergistic rather than additive (19,20). Thus, for both translation and degradation of mRNA, elements binding to the 5Ј and 3Ј termini act cooperatively and in close proximity.There are two major pathways by which polyadenylated mRNA is degraded in eukaryotic cells, a 5Ј33Ј pathway and a 3Ј35Ј pathway, as well as two specialized routes for aberrant mRNA degradation (21). In both the 5Ј33Ј and 3Ј35Јpathways, shortening of the poly(A) tract initiates mRNA decay. There are several mechanism of deadenylation (21), but one of them involves a poly(A)-specific ribonuclease (22), an enzyme tha...

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Section: Methodsmentioning

confidence: 99%

Differential Inhibition of mRNA Degradation Pathways by Novel Cap Analogs

Grudzien

Kalek

Jemielity

et al. 2006

Journal of Biological Chemistry

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113

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“…GppppG were performed as described previously (Darzynkiewicz et al 1990;Stepinski et al 1995;Jankowska et al1996;Niedzwiecka et al 2007 (Darzynkiewicz et al 1985;Jankowska et al 1993;Zuberek et al 2004;Niedzwiecka et al 2007). …”

Section: Cap Analogsmentioning

confidence: 99%

Cap analog substrates reveal three clades of cap guanine-N2 methyltransferases with distinct methyl acceptor specificities

Benarroch¹,

Jankowska-Anyszka²,

Stępiński³

et al. 2009

RNA

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The Tgs proteins are structurally homologous AdoMet-dependent eukaryal enzymes that methylate the N2 atom of 7-methyl guanosine nucleotides. They have an imputed role in the synthesis of the 2,2,7-trimethylguanosine (TMG) RNA cap. Here we exploit a collection of cap-like substrates to probe the repertoire of three exemplary Tgs enzymes, from mammalian, protozoan, and viral sources, respectively. We find that human Tgs (hTgs1) is a bona fide TMG synthase adept at two separable transmethylation steps: (1) conversion of m 7 G to m 2,7 G, and (2) G nucleotides by hTgs1 and GlaTgs2 underscored the importance of guanine N7 alkylation in providing a key p-cation interaction in the methyl acceptor site. Mimivirus Tgs (MimiTgs) shares with the Giardia homolog the ability to catalyze only a single round of methyl addition at guanine-N2, but is distinguished by its capacity for guanine-N2 methylation in the absence of prior N7 methylation. The relaxed cap specificity of MimiTgs is revealed at alkaline pH. Our findings highlight both stark and subtle differences in acceptor specificity and reaction outcomes among Tgs family members.

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“…Syntheses of mono-and dinucleotide cap analogs were performed as described previously (Darzynkiewicz et al 1985(Darzynkiewicz et al , 1990Jemielity et al 2003;Zuberek et al 2004). The cap analog concentrations were determined spectrophotometrically (Cai et al 1999).…”

Section: Synthesis Of Cap Analogsmentioning

confidence: 99%

Weak binding affinity of human 4EHP for mRNA cap analogs

Zuberek¹,

Kubacka

Jabłonowska³

et al. 2007

RNA

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Ribosome recruitment to the majority of eukaryotic mRNAs is facilitated by the interaction of the cap binding protein, eIF4E, with the mRNA 59 cap structure. eIF4E stimulates translation through its interaction with a scaffolding protein, eIF4G, which helps to recruit the ribosome. Metazoans also contain a homolog of eIF4E, termed 4EHP, which binds the cap structure, but not eIF4G, and thus cannot stimulate translation, but it instead inhibits the translation of only one known, and possibly subset mRNAs. To understand why 4EHP does not inhibit general translation, we studied the binding affinity of 4EHP for cap analogs using two methods: fluorescence titration and stopped-flow measurements. We show that 4EHP binds cap analogs m 7 GpppG and m 7 GTP with 30 and 100 lower affinity than eIF4E. Thus, 4EHP cannot compete with eIF4E for binding to the cap structure of most mRNAs.

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Synthesis, Conformation and Hydrolytic Stability of p¹,p³−Dinucleoside Triphosphates Related to mRNA 5′-cap, and Comparative Kinetic Studies on their Nucleoside and Nucleoside Monophosphate Analogs

Cited by 50 publications

References 28 publications

Differential Inhibition of mRNA Degradation Pathways by Novel Cap Analogs

Differential Inhibition of mRNA Degradation Pathways by Novel Cap Analogs

Cap analog substrates reveal three clades of cap guanine-N2 methyltransferases with distinct methyl acceptor specificities

Weak binding affinity of human 4EHP for mRNA cap analogs

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Synthesis, Conformation and Hydrolytic Stability of p1,p3−Dinucleoside Triphosphates Related to mRNA 5′-cap, and Comparative Kinetic Studies on their Nucleoside and Nucleoside Monophosphate Analogs

Cited by 50 publications

References 28 publications

Differential Inhibition of mRNA Degradation Pathways by Novel Cap Analogs

Differential Inhibition of mRNA Degradation Pathways by Novel Cap Analogs

Cap analog substrates reveal three clades of cap guanine-N2 methyltransferases with distinct methyl acceptor specificities

Weak binding affinity of human 4EHP for mRNA cap analogs

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Resources

About

Synthesis, Conformation and Hydrolytic Stability of p¹,p³−Dinucleoside Triphosphates Related to mRNA 5′-cap, and Comparative Kinetic Studies on their Nucleoside and Nucleoside Monophosphate Analogs