Mammalian Nudt15 hydrolytic and binding activity on methylated guanosine mononucleotides.

The Nudt15 enzyme of the NUDIX protein family is the subject of extensive study due to its action on thiopurine drugs used in the treatment of cancer and inflammatory diseases. In addition to thiopurines, Nudt15 is enzymatically active in vitro on several nucleotide substrates. It has also been suggested that this enzyme may play a role in 5'RNA turnover by hydrolyzing m7GDP, a product of mRNA decapping. However, no detailed studies on this substrate with Nudt15 are available. Here, we analyzed the enzymatic activity of Nudt15 with m7GDP, its triphosphate form m7GTP, and the trimethylated counterparts (m32,2,7GDP and m32,2,7GTP). Kinetic data revealed a moderate activity of Nudt15 toward these methylated mononucleotides compared to the dGTP substrate. However m7GDP and m32,2,7GDP showed a distinct stabilization of Nudt15 upon ligand binding, in the same range as dGTP, and thus these two mononucleotides may be used as leading structures in the design of small molecule binders of Nudt15.

Contribution of Nudt12 enzyme to differentially methylated dinucleotides of 5'RNA cap structure.

Recent findings have substantially broadened our knowledge about the diversity of modifications of the 5'end of RNAs, an issue generally attributed to mRNA cap structure (m7GpppN). Nudt12 is one of the recently described new enzymatic activities involved in cap metabolism. However, in contrast to its roles in metabolite-cap turnover (e.g., NAD-cap) and NADH/NAD metabolite hydrolysis, little is known regarding its hydrolytic activity towards dinucleotide cap structures. In order to gain further insight into this Nudt12 activity, comprehensive analysis with a spectrum of cap-like dinucleotides was performed with respect to different nucleotide types adjacent to the (m7)G moiety and its methylation status. Among the tested compounds, GpppA, GpppAm, and Gpppm6Am were identified as novel potent Nudt12 substrates, with KM values in the same range as that of NADH. Interestingly, substrate inhibition of Nudt12 catalytic activity was detected in the case of the GpppG dinucleotide, a phenomenon not reported to date. Finally, comparison of Nudt12 with DcpS and Nud16, two other enzymes with known activity on dinucleotide cap structures, revealed their overlapping and more specific substrates. Altogether, these findings provide a basis for clarifying the role of Nudt12 in cap-like dinucleotide turnover.

Insight into the Binding and Hydrolytic Preferences of hNudt16 Based on Nucleotide Diphosphate Substrates.

Nudt16 is a member of the NUDIX family of hydrolases that show specificity towards substrates consisting of a nucleoside diphosphate linked to another moiety X. Several substrates for hNudt16 and various possible biological functions have been reported. However, some of these reports contradict each other and studies comparing the substrate specificity of the hNudt16 protein are limited. Therefore, we quantitatively compared the affinity of hNudt16 towards a set of previously published substrates, as well as identified novel potential substrates. Here, we show that hNudt16 has the highest affinity towards IDP and GppG, with Kd below 100 nM. Other tested ligands exhibited a weaker affinity of several orders of magnitude. Among the investigated compounds, only IDP, GppG, m7GppG, AppA, dpCoA, and NADH were hydrolyzed by hNudt16 with a strong substrate preference for inosine or guanosine containing compounds. A new identified substrate for hNudt16, GppG, which binds the enzyme with an affinity comparable to that of IDP, suggests another potential regulatory role of this protein. Molecular docking of hNudt16-ligand binding inside the hNudt16 pocket revealed two binding modes for representative substrates. Nucleobase stabilization by Π stacking interactions with His24 has been associated with strong binding of hNudt16 substrates.

Effect of the His-Tag Location on Decapping Scavenger Enzymes and Their Hydrolytic Activity toward Cap Analogs.

Decapping scavenger enzymes (DcpSs) are important players in mRNA degradation machinery and conserved in eukaryotes. Importantly, human DcpS is the recognized target for spinal muscular atrophy (SMA) and acute myeloid leukemia (AML) therapy, and has recently been connected to development of intellectual disability. Most recombinant DcpSs used in biochemical and biophysical studies are prepared as tagged proteins, with polyhistidine (His-tag) at the N-terminus or C-terminus. Our work is the first report on the parallel characterization of three versions of DcpSs (native and N- or C-terminally tagged) of three species (humans, Caenorhabditis elegans , and Ascaris suum). The native forms of all three enzymes were prepared by N-(His)10 tag cleavage. Protein thermal stability, measured by differential scanning fluorimetry (DSF), was unaffected in the case of native and tagged versions of human and A. suum DcpS; however, the melting temperature (T m) of C. elagans DcpS of was significantly influenced by the presence of the additional N- or C-tag. To investigate the impact of the tag positioning on the catalytic properties of DcpS, we tested the hydrolytic activity of native DcpS and their His-tagged counterparts toward cap dinucleotides (m7GpppG and m3 2,2,7GpppG) and m7GDP. The kinetic data indicate that dinucleotide substrates are hydrolyzed with comparable efficiency by native human and A. suum DcpS and their His-tagged forms. In contrast, both His-tagged C. elegans DcpSs exhibited higher activity toward m7GpppG than the native enzyme. m7GDP is resistant to enzymatic cleavage by all three forms of human and nematode DcpS.

Hydrolytic activity of human Nudt16 enzyme on dinucleotide cap analogs and short capped oligonucleotides.

Human Nudt16 (hNudt16) is a member of the Nudix family of hydrolases, comprising enzymes catabolizing various substrates including canonical (d)NTPs, oxidized (d)NTPs, nonnucleoside polyphosphates, and capped mRNAs. Decapping activity of the Xenopus laevis (X29) Nudt16 homolog was observed in the nucleolus, with a high specificity toward U8 snoRNA. Subsequent studies have reported cytoplasmic localization of mammalian Nudt16 with cap hydrolysis activity initiating RNA turnover, similar to Dcp2. The present study focuses on hNudt16 and its hydrolytic activity toward dinucleotide cap analogs and short capped oligonucleotides. We performed a screening assay for potential dinucleotide and oligonucleotide substrates for hNudt16. Our data indicate that dinucleotide cap analogs and capped oligonucleotides containing guanine base in the first transcribed nucleotide are more susceptible to enzymatic digestion by hNudt16 than their counterparts containing adenine. Furthermore, unmethylated dinucleotides (GpppG and ApppG) and respective oligonucleotides (GpppG-16nt and GpppA-16nt) were hydrolyzed by hNudt16 with greater efficiency than were m7GpppG and m7GpppG-16nt. In conclusion, we found that hNudt16 hydrolysis of dinucleotide cap analogs and short capped oligonucleotides displayed a broader spectrum specificity than is currently known.

Molecular recognition of mRNA 5' cap by 3' poly(A)-specific ribonuclease (PARN) differs from interactions known for other cap-binding proteins.

The mRNA 5' cap structure plays a pivotal role in coordination of eukaryotic translation and mRNA degradation. Poly(A)-specific ribonuclease (PARN) is a dimeric exoribonuclease that efficiently degrades mRNA 3' poly(A) tails while also simultaneously interacting with the mRNA 5' cap. The cap binding amplifies the processivity of PARN action. We used surface plasmon resonance kinetic analysis, quantitative equilibrium fluorescence titrations and circular dichroism to study the cap binding properties of PARN. The molecular mechanism of 5' cap recognition by PARN has been demonstrated to differ from interactions seen for other known cap-binding proteins in that: i) the auxiliary biological function of 5' cap binding by the 3' degrading enzyme is accomplished by negative cooperativity of PARN dimer subunits; ii) non-coulombic interactions are major factors in the complex formation; and iii) PARN has versatile activity toward alternative forms of the cap. These characteristics contribute to stabilization of the PARN-cap complex needed for the deadenylation processivity. Our studies provide a consistent biophysical basis for elucidation of the processive mechanism of PARN-mediated 3' mRNA deadenylation and provide a new framework to interpret the role of the 5' cap in mRNA degradation.

Effect of different N7 substitution of dinucleotide cap analogs on the hydrolytic susceptibility towards scavenger decapping enzymes (DcpS).

Scavenger decapping enzymes (DcpS) are involved in eukaryotic mRNA degradation process. They catalyze the cleavage of residual cap structure m(7)GpppN and/or short capped oligonucleotides resulting from exosom-mediated the 3' to 5' digestion. For the specific cap recognition and efficient degradation by DcpS, the positive charge at N7 position of guanine moiety is required. Here we examine the role the N7 substitution within the cap structure on the interactions with DcpS (human, Caenorhabditis elegans and Ascaris suum) comparing the hydrolysis rates of dinucleotide cap analogs (m(7)GpppG, et(7)GpppG, but(7)GpppG, bn(7)GpppG) and the binding affinities of hydrolysis products (m(7)GMP, et(7)GMP, but(7)GMP, bn(7)GMP). Our results show the conformational flexibility of the region within DcpS cap-binding pocket involved in the interaction with N7 substituted guanine, which enables accommodation of substrates with differently sized N7 substituents.

eIF4F-like complexes formed by cap-binding homolog TbEIF4E5 with TbEIF4G1 or TbEIF4G2 are implicated in post-transcriptional regulation in Trypanosoma brucei.

Members of the eIF4E mRNA cap-binding family are involved in translation and the modulation of transcript availability in other systems as part of a three-component complex including eIF4G and eIF4A. The kinetoplastids possess four described eIF4E and five eIF4G homologs. We have identified two new eIF4E family proteins in Trypanosoma brucei, and define distinct complexes associated with the fifth member, TbEIF4E5. The cytosolic TbEIF4E5 protein binds cap 0 in vitro. TbEIF4E5 was found in association with two of the five TbEIF4Gs. TbIF4EG1 bound TbEIF4E5, a 47.5-kDa protein with two RNA-binding domains, and either the regulatory protein 14-3-3 II or a 117.5-kDa protein with guanylyltransferase and methyltransferase domains in a potentially dynamic interaction. The TbEIF4G2/TbEIF4E5 complex was associated with a 17.9-kDa hypothetical protein and both 14-3-3 variants I and II. Knockdown of TbEIF4E5 resulted in the loss of productive cell movement, as evidenced by the inability of the cells to remain in suspension in liquid culture and the loss of social motility on semisolid plating medium, as well as a minor reduction of translation. Cells appeared lethargic, as opposed to compromised in flagellar function per se. The minimal use of transcriptional control in kinetoplastids requires these organisms to implement downstream mechanisms to regulate gene expression, and the TbEIF4E5/TbEIF4G1/117.5-kDa complex in particular may be a key player in that process. We suggest that a pathway involved in cell motility is affected, directly or indirectly, by one of the TbEIF4E5 complexes.

Endocardial silicone lead wear: description of tribological phenomena on the basis of microscopic examination of removed leads. Preliminary report.

BACKGROUND: The passage of a lead in tissues and in the cardiovascular system depends on the implantation technique. The structure of the leads, which is a combination of two or more materials, triggers their wear. Breakage of the external pacemaker (PM) lead insulation causes unsealing of the lead and exposure of its internal spaces, which can be the anchor of lead-dependent infective endocarditis (LDIE). In the case of implantable cardioverter-defibrillator (ICD) leads, damage to external insulation isthe cause of externalisation of the cable. AIM: To describe endocardial lead abrasion as a tribological phenomenon resulting from rubbing the leads against each other in the mechanism of polymer on polymer friction, and other mechanisms associated with lead structure i.e. polymer on metal friction. METHODS: Twenty-two leads were extracted from ten patients (three women) aged 66.5 ± 13.4 years. In all cases, the reason for lead removal was infection ­ in 80% LDIE. The PM (one ICD) two- and three-lead systems, all with silicone insulation, were aged 3­25, mean 8.3 years. The destroyed polymer insulation was examined by optical and scanning electron microscopy. The site of damage was defined as the length of the lead from its distal end. This lead segment motion was analysed on chest scopy performed prior to the removal procedure. In this way, three sites of lead damage were distinguished: intracardiac, intravenous, and intrapocket. Tribological wear was observed on the polymer-metal interface and between the leads. The following characteristics were recorded: the type of PM or ICD system in which the extracted leads worked, the lead dwell time,and the lead model. RESULTS: Scanning electron examinations showed that in all cases lead insulation had undergone tribological failure. In all samples, the image of fatigue wear was recorded. In all examined places, we found evidence that adhesive wear was present with the transfer of material to the edges of friction zones and/or to friction partners. In 80% of the patients with LDIE, a total breakage of insulation and abrasive wear was observed, especially when a lead cyclically bent and rubbed against another lead. Abrasive wear was the cause of lead unsealing at sites of strong lead bending, in the right atrium near the tricuspid valve. CONCLUSIONS: Acknowledging the tribological mechanism may connect the commonly known crush syndrome with lead abrasion in the cardiac implantable device pocket and in the heart cavity.

Structural analysis of human 2'-O-ribose methyltransferases involved in mRNA cap structure formation.

The 5' cap of human messenger RNA contains 2'-O-methylation of the first and often second transcribed nucleotide that is important for its processing, translation and stability. Human enzymes that methylate these nucleotides, termed CMTr1 and CMTr2, respectively, have recently been identified. However, the structures of these enzymes and their mechanisms of action remain unknown. In the present study, we solve the crystal structures of the active CMTr1 catalytic domain in complex with a methyl group donor and a capped oligoribonucleotide, thereby revealing the mechanism of specific recognition of capped RNA. This mechanism differs significantly from viral enzymes, thus providing a framework for their specific targeting. Based on the crystal structure of CMTr1, a comparative model of the CMTr2 catalytic domain is generated. This model, together with mutational analysis, leads to the identification of residues involved in RNA and methyl group donor binding.

7-methylguanosine diphosphate (m(7)GDP) is not hydrolyzed but strongly bound by decapping scavenger (DcpS) enzymes and potently inhibits their activity.

Decapping scavenger (DcpS) enzymes catalyze the cleavage of a residual cap structure following 3' → 5' mRNA decay. Some previous studies suggested that both m(7)GpppG and m(7)GDP were substrates for DcpS hydrolysis. Herein, we show that mononucleoside diphosphates, m(7)GDP (7-methylguanosine diphosphate) and m(3)(2,2,7)GDP (2,2,7-trimethylguanosine diphosphate), resulting from mRNA decapping by the Dcp1/2 complex in the 5' → 3' mRNA decay, are not degraded by recombinant DcpS proteins (human, nematode, and yeast). Furthermore, whereas mononucleoside diphosphates (m(7)GDP and m(3)(2,2,7)GDP) are not hydrolyzed by DcpS, mononucleoside triphosphates (m(7)GTP and m(3)(2,2,7)GTP) are, demonstrating the importance of a triphosphate chain for DcpS hydrolytic activity. m(7)GTP and m(3)(2,2,7)GTP are cleaved at a slower rate than their corresponding dinucleotides (m(7)GpppG and m(3)(2,2,7)GpppG, respectively), indicating an involvement of the second nucleoside for efficient DcpS-mediated digestion. Although DcpS enzymes cannot hydrolyze m(7)GDP, they have a high binding affinity for m(7)GDP and m(7)GDP potently inhibits DcpS hydrolysis of m(7)GpppG, suggesting that m(7)GDP may function as an efficient DcpS inhibitor. Our data have important implications for the regulatory role of m(7)GDP in mRNA metabolic pathways due to its possible interactions with different cap-binding proteins, such as DcpS or eIF4E.

Structural basis for nematode eIF4E binding an m(2,2,7)G-Cap and its implications for translation initiation.

Metazoan spliced leader (SL) trans-splicing generates mRNAs with an m(2,2,7)G-cap and a common downstream SL RNA sequence. The mechanism for eIF4E binding an m²²7G-cap is unknown. Here, we describe the first structure of an eIF4E with an m(2,2,7)G-cap and compare it to the cognate m7G-eIF4E complex. These structures and Nuclear Magnetic Resonance (NMR) data indicate that the nematode Ascaris suum eIF4E binds the two different caps in a similar manner except for the loss of a single hydrogen bond on binding the m(2,2,7)G-cap. Nematode and mammalian eIF4E both have a low affinity for m(2,2,7)G-cap compared with the m7G-cap. Nematode eIF4E binding to the m7G-cap, m(2,2,7)G-cap and the m(2,2,7)G-SL 22-nt RNA leads to distinct eIF4E conformational changes. Additional interactions occur between Ascaris eIF4E and the SL on binding the m(2,2,7)G-SL. We propose interactions between Ascaris eIF4E and the SL impact eIF4G and contribute to translation initiation, whereas these interactions do not occur when only the m(2,2,7)G-cap is present. These data have implications for the contribution of 5'-UTRs in mRNA translation and the function of different eIF4E isoforms.

Thermodynamics of molecular recognition of mRNA 5' cap by yeast eukaryotic initiation factor 4E.

Molecular mechanisms underlying the recognition of the mRNA 5' terminal structure called "cap" by the eukaryotic initiation factor 4E (eIF4E) are crucial for cap-dependent translation. To gain a deeper insight into how the yeast eIF4E interacts with the cap structure, isothermal titration calorimetry and the van't Hoff analysis based on intrinsic protein fluorescence quenching upon titration with a series of chemical cap analogs were performed, providing a consistent thermodynamic description of the binding process in solution. Equilibrium association constants together with thermodynamic parameters revealed similarities and differences between yeast and mammalian eIF4Es. The yeast eIF4E complex formation was enthalpy-driven and entropy-opposed for each cap analog at 293 K. A nontrivial isothermal enthalpy­entropy compensation was found, described by a compensation temperature, T(c) = 411 ± 18 K. For a low affinity analog, 7-methylguanosine monophosphate, a heat capacity change was detected, ΔC(p)° = +5.2 ± 1.3 kJ·mol(-1)·K(-1). The charge-related interactions involving the 5'-5' triphosphate bridge of the cap and basic amino acid side chains at the yeast eIF4E cap-binding site were significantly weaker (by ΔΔH°(vH) of about +10 kJ·mol(-1)) than those for the mammalian homologues, suggesting their optimization during the evolution.

Structural analysis of 5'-mRNA-cap interactions with the human AGO2 MID domain.

In RNA silencing, microRNA (miRNA)-mediated translational repression occurs through mechanisms that do not invoke messenger-RNA (mRNA) target cleavage by Argonaute proteins. The nature of these mechanisms is unclear, but several recent studies have proposed that a direct interaction between the mRNA-cap and the middle (MID) domain of Argonautes is involved. Here, we present crystallographic and NMR data demonstrating that cap analogues do not bind significantly to the isolated MID domain of human Argonaute 2 (hAGO2) and are found in the miRNA 5'-nucleotide binding site in an implausible binding mode. Additionally, in vitro pull-down experiments with full-length hAGO2 indicate that the interaction with cap analogues is nonspecific.

Plant nucleoside 5'-phosphoramidate hydrolase; simple purification from yellow lupin (Lupinus luteus) seeds and properties of homogeneous enzyme.

Adenosine 5'-phosphoramidate (NH2-pA) is an uncommon natural nucleotide of poorly understood biochemistry and function. We studied a plant enzyme potentially involved in the catabolism of NH2-pA. A fast and simple method comprising extraction of yellow lupin (Lupinus luteus) seed-meal with a low ionic strength buffer, ammonium sulfate and acetone fractionations, removal of contaminating proteins by heat denaturation, and affinity chromatography on AMP-agarose, yielded homogenous nucleoside 5'-phosphoramidase. Mass spectrometric analysis showed that the lupin hydrolase exhibits closest similarity to Arabidopsis thaliana Hint1 protein. The substrate specificity of the lupin enzyme, in particular its ability to split the P-S bond in adenosine 5'-phosphorothioate, is typical of known Hint1 proteins. Adenosine 5'-phosphofluoride and various derivatives of guanosine 5'-phosphoramidate were also substrates. Neither common divalent metal cations nor 10 mM EDTA or EGTA affected the hydrolysis of NH2-pA. The enzyme functions as a homodimer (2 x 15,800 Da). At the optimum pH of 7.0, the K(m) for NH2-pA was 0.5 µM and k(cat) 0.8 s⁻¹ (per monomer active site). The properties of the lupin nucleoside 5'-phosphoramidase are compared with those of its counterparts from other organisms.

Characterization of hMTr1, a human Cap1 2'-O-ribose methyltransferase.

Cellular eukaryotic mRNAs are capped at their 5' ends with a 7-methylguanosine nucleotide, a structural feature that has been shown to be important for conferring mRNA stability, stimulating mRNA biogenesis (splicing, poly(A) addition, nucleocytoplasmic transport), and increasing translational efficiency. Whereas yeast mRNAs have no additional modifications to the cap, called cap0, higher eukaryotes are methylated at the 2'-O-ribose of the first or the first and second transcribed nucleotides, called cap1 and cap2, respectively. In the present study, we identify the methyltransferase responsible for cap1 formation in human cells, which we call hMTr1 (also known as FTSJD2 and ISG95). We show in vitro that hMTr1 catalyzes specific methylation of the 2'-O-ribose of the first nucleotide of a capped RNA transcript. Using siRNA-mediated knockdown of hMTr1 in HeLa cells, we demonstrate that hMTr1 is responsible for cap1 formation in vivo.

Structural requirements for Caenorhabditis elegans DcpS substrates based on fluorescence and HPLC enzyme kinetic studies.

The activity of the Caenorhabditis elegans scavenger decapping enzyme (DcpS) on its natural substrates and dinucleotide cap analogs, modified with regard to the nucleoside base or ribose moiety, has been examined. All tested dinucleotides were specifically cleaved between beta- and gamma-phosphate groups in the triphosphate chain. The kinetic parameters of enzymatic hydrolysis (K(m), V(max)) were determined using fluorescence and HPLC methods, as complementary approaches for the kinetic studies of C. elegans DcpS. From the kinetic data, we determined which parts of the cap structure are crucial for DcpS binding and hydrolysis. We showed that m(3)(2,2,7)GpppG and m(3)(2,2,7)GpppA are cleaved with higher rates than their monomethylated counterparts. However, the higher specificity of C. elegans DcpS for monomethylguanosine caps is illustrated by the lower K(m) values. Modifications of the first transcribed nucleotide did not affect the activity, regardless of the type of purine base. Our findings suggest C. elegans DcpS flexibility in the first transcribed nucleoside-binding pocket. Moreover, although C. elegans DcpS accommodates bulkier groups in the N7 position (ethyl or benzyl) of the cap, both 2'-O- and 3'-O-methylations of 7-methylguanosine result in a reduction in hydrolysis by two orders of magnitude.

The nematode eukaryotic translation initiation factor 4E/G complex works with a trans-spliced leader stem-loop to enable efficient translation of trimethylguanosine-capped RNAs.

Eukaryotic mRNA translation begins with recruitment of the 40S ribosome complex to the mRNA 5' end through the eIF4F initiation complex binding to the 5' m(7)G-mRNA cap. Spliced leader (SL) RNA trans splicing adds a trimethylguanosine (TMG) cap and a sequence, the SL, to the 5' end of mRNAs. Efficient translation of TMG-capped mRNAs in nematodes requires the SL sequence. Here we define a core set of nucleotides and a stem-loop within the 22-nucleotide nematode SL that stimulate translation of mRNAs with a TMG cap. The structure and core nucleotides are conserved in other nematode SLs and correspond to regions of SL1 required for early Caenorhabditis elegans development. These SL elements do not facilitate translation of m(7)G-capped RNAs in nematodes or TMG-capped mRNAs in mammalian or plant translation systems. Similar stem-loop structures in phylogenetically diverse SLs are predicted. We show that the nematode eukaryotic translation initiation factor 4E/G (eIF4E/G) complex enables efficient translation of the TMG-SL RNAs in diverse in vitro translation systems. TMG-capped mRNA translation is determined by eIF4E/G interaction with the cap and the SL RNA, although the SL does not increase the affinity of eIF4E/G for capped RNA. These results suggest that the mRNA 5' untranslated region (UTR) can play a positive and novel role in translation initiation through interaction with the eIF4E/G complex in nematodes and raise the issue of whether eIF4E/G-RNA interactions play a role in the translation of other eukaryotic mRNAs.

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

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) conversion of m(2,7)G to m(2,2,7)G. hTgs1 is unable to methylate G or m(2)G, signifying that both steps require an m(7)G cap. hTgs1 utilizes a broad range of m(7)G nucleotides, including mono-, di-, tri-, and tetraphosphate derivatives as well as cap dinucleotides with triphosphate or tetraphosphate bridges. In contrast, Giardia lamblia Tgs (GlaTgs2) exemplifies a different clade of guanine-N2 methyltransferase that synthesizes only a dimethylguanosine (DMG) cap structure and cannot per se convert DMG to TMG under any conditions tested. Methylation of benzyl(7)G and ethyl(7)G nucleotides by hTgs1 and GlaTgs2 underscored the importance of guanine N7 alkylation in providing a key pi-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.

Structural insights into parasite eIF4E binding specificity for m7G and m2,2,7G mRNA caps.

The eukaryotic translation initiation factor eIF4E recognizes the mRNA cap, a key step in translation initiation. Here we have characterized eIF4E from the human parasite Schistosoma mansoni. Schistosome mRNAs have either the typical monomethylguanosine (m(7)G) or a trimethylguanosine (m(2,2,7)G) cap derived from spliced leader trans-splicing. Quantitative fluorescence titration analyses demonstrated that schistosome eIF4E has similar binding specificity for both caps. We present the first crystal structure of an eIF4E with similar binding specificity for m(7)G and m(2,2,7)G caps. The eIF4E.m(7)GpppG structure demonstrates that the schistosome protein binds monomethyl cap in a manner similar to that of single specificity eIF4Es and exhibits a structure similar to other known eIF4Es. The structure suggests an alternate orientation of a conserved, key Glu-90 in the cap-binding pocket that may contribute to dual binding specificity and a position for mRNA bound to eIF4E consistent with biochemical data. Comparison of NMR chemical shift perturbations in schistosome eIF4E on binding m(7)GpppG and m(2,2,7)GpppG identified key differences between the two complexes. Isothermal titration calorimetry demonstrated significant thermodynamics differences for the binding process with the two caps (m(7)G versus m(2,2,7)G). Overall the NMR and isothermal titration calorimetry data suggest the importance of intrinsic conformational flexibility in the schistosome eIF4E that enables binding to m(2,2,7)G cap.