Structural Basis for Sequence-Nonspecific Recognition of 5′-Capped mRNA by a Cap-Modifying Enzyme

Hodel, A.E.; Gershon, Paul D.; Quiocho, Florante A.

doi:10.1016/s1097-2765(00)80044-1

Cited by 172 publications

(205 citation statements)

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“…Other non-eIF4E capbinding proteins, e.g., CBC (4) and VP39 (26), bind the cap via cation-π or π-π stacking arrangements using two aromatic residues similarly to other eIF4E family members. Indeed, there are only two examples of specific cap-binding proteins that chelate cap via a strategy different to the aromatic sandwich.…”

Section: Resultsmentioning

confidence: 99%

eIF4E3 acts as a tumor suppressor by utilizing an atypical mode of methyl-7-guanosine cap recognition

Osborne

Volpon

Kornblatt

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

104

143

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Recognition of the methyl-7-guanosine (m 7 G) cap structure on mRNA is an essential feature of mRNA metabolism and thus gene expression. Eukaryotic translation initiation factor 4E (eIF4E) promotes translation, mRNA export, proliferation, and oncogenic transformation dependent on this cap-binding activity. eIF4E-cap recognition is mediated via complementary charge interactions of the positively charged m 7 G cap between the negative π-electron clouds from two aromatic residues. Here, we demonstrate that a variant subfamily, eIF4E3, specifically binds the m 7 G cap in the absence of an aromatic sandwich, using instead a different spatial arrangement of residues to provide the necessary electrostatic and van der Waals contacts. Contacts are much more extensive between eIF4E3-cap than other family members. Structural analyses of other cap-binding proteins indicate this recognition mode is atypical. We demonstrate that eIF4E3 relies on this cap-binding activity to act as a tumor suppressor, competing with the growthpromoting functions of eIF4E. In fact, reduced eIF4E3 in high eIF4E cancers suggests that eIF4E3 underlies a clinically relevant inhibitory mechanism that is lost in some malignancies. Taken together, there is more structural plasticity in cap recognition than previously thought, and this is physiologically relevant.M etabolism of mRNA is a complex and highly regulated process dependent on the association of the methyl-7-guanosine (m 7 G) cap structure on the 5′ end of transcripts with appropriate proteins (1-3). In mammalian cells, the two major cap-binding proteins are the nuclear cap-binding complex (CBC) (4) and eukaryotic translation initiation factor 4E (eIF4E) (3). Specific cap recognition is key for the fate of transcripts and impacts processes such as mRNA processing and bulk mRNA export via the CBC or the nuclear export of specific transcripts as well as bulk translation by eIF4E (5). NMR and crystallographic studies reveal that m 7 G cap is specifically recognized by both the CBC and eIF4E via intercalation of the m 7 G cap moiety with the side chains of two aromatic residues, i.e., an aromatic sandwich (4, 6-9). This is an electrostatically driven process relying on the partial positive charge of the m 7 G cap and the negative π-electron clouds of the aromatic residues. The aromatic residues are completely conserved in these cap-binding proteins. This recognition motif is used almost exclusively by proteins that specifically bind the m 7 G cap including eIF4E, CBC, and vaccinia virus protein VP39 (4, 10).Dysregulation of cap-binding proteins can have striking physiological consequences. For instance through its cap-binding activity and subsequent effects on gene expression, eIF4E plays an important role in proliferation and survival (1, 3). Indeed, eIF4E is overexpressed in about 30% of human cancers and its overexpression is oncogenic in cell culture and animal models (2, 11). Mutation of the cap-binding site of eIF4E impairs its activities in translation, mRNA export, and oncogenic transform...

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Section: Resultsmentioning

confidence: 99%

eIF4E3 acts as a tumor suppressor by utilizing an atypical mode of methyl-7-guanosine cap recognition

Osborne

Volpon

Kornblatt

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

104

143

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“…The methyl group at the N7 position does not influence the 29O-MTase activity of NS5MTase DV , indicating that the enzyme does not discriminate methylated from nonmethylated RNA cap structures in the 29O-methylation step in contrast to specific viral 29O-MTases (Decroly et al, 2008;Hodel et al, 1998). The NS5MTase DV 29O-MTase activity showed steady-state parameters of both substrates and characteristics that are in agreement with other AdoMet-dependent MTases acting on polynucleotide substrates and in particular with viral 29O-MTases.…”

Section: Resultsmentioning

confidence: 99%

“…The presence of the reducing agent dithiothreitol (DTT; 5 mM) stabilized NS5MTase DV during long time-course experiments (data not shown). In all subsequent tests of NS5MTase DV on 7Me GpppAC n and GpppAC n , we used 40 mM Tris/HCl (pH 7.5) and 5 mM DTT in the absence of MgCl 2 .Interestingly, a similar bell-shaped pH curve with an optimum at 7.5 and independence from divalent cations was found for vaccinia virus mRNA cap 29O-MTase VP39 (Barbosa & Moss, 1978) containing the same catalytic tetrad, K-D-K-E (Hodel et al, 1996(Hodel et al, , 1998. Nevertheless, the optimum conditions for NS5MTase DV described here are in contrast to a reported pH optimum of 29O-MTase activity of NS5MTase DV on another type of short RNA substrate, GpppAGAACCUG, reported recently (Kroschewski et al, 2008;Lim et al, 2008).…”

mentioning

confidence: 89%

“…The cap G is bound to the cap-binding site (taken from a previous model of GpppA in a 29O-methylation model; Egloff et al, 2007). The remaining AGACUA was taken from a complex structure of DV helicase (Luo et al, 2008) and placed using the capped RNA in the complex of mRNA cap 29O-MTase VP39 (Hodel et al, 1998) as an orientation. The co-factor AdoMet (from the model in Egloff et al, 2007) is bound to its binding site on the right site of the active site and is partly hidden by a protruding loop.…”

Section: Michaelis-menten Parametersmentioning

confidence: 99%

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Biochemical characterization of the (nucleoside-2'O)-methyltransferase activity of dengue virus protein NS5 using purified capped RNA oligonucleotides 7MeGpppACn and GpppACn

Selisko

Peyrane

Canard

et al. 2009

Journal of General Virology

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The flavivirus RNA genome contains a conserved cap-1 structure, 7Me GpppA 29OMe G, at the 59 end. Two mRNA cap methyltransferase (MTase) activities involved in the formation of the cap, the (guanine-N7)-and the (nucleoside-29O)-MTases (29O-MTase), reside in a single domain of nonstructural protein NS5 (NS5MTase). This study reports on the biochemical characterization of the 29O-MTase activity of NS5MTase of dengue virus (NS5MTase DV ) using purified, short, capped RNA substrates ( 7Me GpppAC n or GpppAC n ). NS5MTase DV methylated both types of substrate exclusively at the 29O position. The efficiency of 29O-methylation did not depend on the methylation of the N7 position. Using 7Me GpppAC n and GpppAC n substrates of increasing chain lengths, it was found that both NS5MTase DV 29O activity and substrate binding increased before reaching a plateau at n55. Thus, the cap and 6 nt might define the interface providing efficient binding of enzyme and substrate. K m values for 7Me GpppAC 5 and the co-substrate S-adenosyl-Lmethionine (AdoMet) were determined (0.39 and 3.26 mM, respectively). As reported for other AdoMet-dependent RNA and DNA MTases, the 29O-MTase activity of NS5MTase DV showed a low turnover of 3.25¾10 "4 s "1 . Finally, an inhibition assay was set up and tested on GTP and AdoMet analogues as putative inhibitors of NS5MTase DV , which confirmed efficient inhibition by the reaction product S-adenosyl-homocysteine (IC 50 0.34 mM) and sinefungin (IC 50 0.63 mM), demonstrating that the assay is sufficiently sensitive to conduct inhibitor screening and characterization assays. INTRODUCTIONMost eukaryotic and viral mRNAs are modified by the addition of a cap structure at the 59 end. The mRNA cap consists of an N7-methylguanosine linked to the first transcribed nucleotide by a 59-59 triphosphate bridge (cap-0 structure, 7Me GpppN) (Shuman, 2001). Methylation of the 29O positions of the ribose of the first or the second transcribed nucleotide leads to a cap-1 ( 7Me GpppN 29OMe ) or cap-2 ( 7Me GpppN 29OMe N 29OMe ) structure, respectively. The cap structure stabilizes mRNA and plays a vital role in its translation by controlling mRNA splicing, its transport across the nuclear membrane and its recognition by the eIF4E translation factor (Furuichi & Shatkin, 2000). In eukaryotic cells, the acquisition of the cap-0 structure is a co-transcriptional event. RNA capping implies three steps in which the 59 triphosphate end of RNA is hydrolysed to a 59 diphosphate by an RNA triphosphatase (RTPase), then capped with GMP by a guanylyltransferase (GTase) and finally methylated at the N7 position of the guanine by an S-adenosyl-L-methionine (AdoMet)-dependent (guanine-N7)-methyltransferase (N7-MTase) (Gu & Lima, 2005). In contrast to mRNAs of lower eukaryotes, which bear a cap-0 structure, the mRNAs of higher eukaryotes acquire a cap-1 structure via the action of a (nucleoside-29O)-MTase (29O-MTase) (Langberg & Moss, 1981). Compared with N7 methylation, this mRNA methylation step seems to be less important for binding, tr...

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“…The failure of VP39 to catalyze the 2Ј-O-methylation of capped RNAs whose cap nucleotide contains an unmethylated guanine (4), makes the enzyme particularly suitable for investigating the specific recognition of an alkylated, positively charged base. Crystallographic studies have also revealed no binding of unmethylated nucleosides and nucleotides (5,6).…”

mentioning

confidence: 99%

Insertion of an N7-methylguanine mRNA Cap between Two Coplanar Aromatic Residues of a Cap-binding Protein Is Fast and Selective for a Positively Charged Cap

Tsai

2003

Journal of Biological Chemistry

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The N7-methylated guanosine (m 7 G) cap structure, which is found at the 5 ends of mature eukaryotic mRNAs, is critical to a myriad of biological processes. The twenty structures of complexes of cap nucleosides and nucleotides and methylated bases with the vaccinia virus VP39, a cap-specific RNA 2-O-methyltransferase, which we have determined previously, have revealed the atomic basis of cap binding. The precise insertion and tight fitting of the m 7 Gua moiety of the cap between two parallel aromatic residues that are spaced only 6.8 Å apart governs the high specificity of binding. Here we report the investigation of the reaction mechanism of VP39 with three capped ligands (m 7 G, m 7 GpppG, and m 7 GpppGA 3 ) by fluorescence stopped-flow technique. Cap binding is a simple one-step mechanism with very fast association rate constant (ϳ10 7 M ؊1 s ؊1 ). Moreover, the pH dependence on the association rate constant of m 7 G binding indicates that only the positively charged keto tautomer of the cap is recognized and bound. The association and dissociation rate constants and affinity constants of the three ligands do not vary greatly, demonstrating that binding is achieved almost entirely by the interactions of m 7 Gua with two aromatic residues in a cation-sandwich.

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Structural Basis for Sequence-Nonspecific Recognition of 5′-Capped mRNA by a Cap-Modifying Enzyme

Cited by 172 publications

References 22 publications

eIF4E3 acts as a tumor suppressor by utilizing an atypical mode of methyl-7-guanosine cap recognition

eIF4E3 acts as a tumor suppressor by utilizing an atypical mode of methyl-7-guanosine cap recognition

Biochemical characterization of the (nucleoside-2'O)-methyltransferase activity of dengue virus protein NS5 using purified capped RNA oligonucleotides 7MeGpppACn and GpppACn

Insertion of an N7-methylguanine mRNA Cap between Two Coplanar Aromatic Residues of a Cap-binding Protein Is Fast and Selective for a Positively Charged Cap

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