Marzena Jankowska-Anyszka

Multiple Isoforms of Eukaryotic Protein Synthesis Initiation Factor 4E in Caenorhabditis elegans Can Distinguish between Mono- and Trimethylated mRNA Cap Structures

The rate-limiting step for cap-dependent translation initiation in eukaryotes is recruitment of mRNA to the ribosome. An early event in this process is recognition of the m7GTP-containing cap structure at the 5′-end of the mRNA by initiation factor eIF4E. In the nematode Caenorhabditis elegans, mRNAs from 70% of the genes contain a different cap structure, m3 2,2,7GTP. This cap structure is poorly recognized by mammalian elF4E, suggesting that C. elegansmay possess a specialized form of elF4E that can recognize m3 2,2,7GTP. Analysis of the C. elegans genomic sequence data base revealed the presence of three elF4E-like genes, here named ife-1, ife-2, andife-3. cDNAs for these three eIF4E isoforms were cloned and sequenced. Isoform-specific antibodies were prepared from synthetic peptides based on nonhomologous regions of the three proteins. All three eIF4E isoforms were detected in extracts of C. elegans and were retained on m7GTP-Sepharose. One eIF4E isoform, IFE-1, was also retained on m3 2,2,7GTP-Sepharose. Furthermore, binding of IFE-1 and IFE-2 to m7GTP-Sepharose was inhibited by m3 2,2,7GTP. These results suggest that IFE-1 and IFE-2 bind both m7GTP- and m3 2,2,7GTP-containing mRNA cap structures, although with different affinities. In conjunction with IFE-3, these eIF4E isoforms would permit cap-dependent recruitment of all C. elegans mRNAs to the ribosome.

Discrimination between mono–and trimethylated cap structures by two isoforms of Caenorhabditis elegans eIF4E

Primitive eukaryotes like Caenorhabditis elegans produce mRNAs capped with either m7GTP or m32,2,7GTP. Caenorhabditis elegans also expresses five isoforms of the cap‐binding protein eIF4E. Some isoforms (e.g. IFE‐3) bind to m7GTP–Sepharose exclusively, whereas others (e.g. IFE‐5) bind to both m7GTP− and m32,2,7GTP–Sepharose. To examine specificity differences, we devised molecular models of the tertiary structures of IFE‐3 and IFE‐5, based on the known structure of mouse eIF4E‐1. We then substituted amino acid sequences of IFE‐5 with homologous sequences from IFE‐3. As few as two changes (N64Y/V65L) converted the cap specificity of IFE‐5 to essentially that of IFE‐3. Molecular dynamics simulations suggested that the width and depth of the cap‐binding cavity were larger in IFE‐5 than in IFE‐3 or the N64Y/V65L variant, supporting a model in which IFE‐3 discriminates against m32,2,7GTP by steric hindrance. Furthermore, the affinity of IFE‐5 (but not IFE‐3) for m32,2,7GTP was reversibly increased when thiol reagents were removed. This was correlated with the formation of a disulfide bond between Cys‐122 and Cys‐126. Thus, translation of m32,2,7GTP‐capped mRNAs may be regulated by intracellular redox state.

Guanosine nucleotide analogs as inhibitors of alphavirus mRNA capping enzyme.

The two virus-specific reactions in the capping of alphavirus RNAs, catalyzed by the replicase protein nsP1, are promising targets for developing virus-specific inhibitors. In this report, we have studied the effect of over 50 cap analogs on the guanine-7-methyltransferase and guanylyltransferase activities of Semliki Forest virus nsP1. Recombinant nsP1 was expressed in Escherichia coli and partially purified by flotation in a discontinuous sucrose gradient. The methyltransferase activity had a pH optimum between pH 6.5 and 7.1, and the apparent Km values were 1.9 mM for GTP, 6.0 microM for S-adenosyl-L-methionine and 170 microM for Mg2+. NsP1 methyltransferase was able to methylate efficiently GTP (relative activity 100%), GDP (16%), GpppG (35%), GppppG (50%) and less efficiently GpppA (12%), m2GTP (9%), and m2,2GTP (25%), but not m7GppG. The most potent inhibitors for nsP1 methyltransferase were et2m7GMP (Ki value 42 microM), m2,7GMP, (64 microM), m2,7GpppG (82 microM), m2et7GMP (105 microM), m2(2-phet)7GMP (194 microM) and m2GMP (386 microM). Of these compounds, m2GMP, m2et7GMP and m2(2-phet)7GMP showed competitive inhibition, whereas the others showed mixed type inhibition. All compounds that inhibited the methyltransferase activity inhibited also the guanylyltransferase activity of nsP1.

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 (m7G) or a trimethylguanosine (m2,2,7G) 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 m7G and m2,2,7G caps. The eIF4E·m7GpppG 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 m7GpppG and m2,2,7GpppG identified key differences between the two complexes. Isothermal titration calorimetry demonstrated significant thermodynamics differences for the binding process with the two caps (m7G versus m2,2,7G). Overall the NMR and isothermal titration calorimetry data suggest the importance of intrinsic conformational flexibility in the schistosome eIF4E that enables binding to m2,2,7G cap.

Structural basis for nematode eIF4E binding an m2,2,7G-Cap and its implications for translation initiation

Metazoan spliced leader (SL) trans-splicing generates mRNAs with an m2,2,7G-cap and a common downstream SL RNA sequence. The mechanism for eIF4E binding an m2,2,7G-cap is unknown. Here, we describe the first structure of an eIF4E with an m2,2,7G-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 m2,2,7G-cap. Nematode and mammalian eIF4E both have a low affinity for m2,2,7G-cap compared with the m7G-cap. Nematode eIF4E binding to the m7G-cap, m2,2,7G-cap and the m2,2,7G-SL 22-nt RNA leads to distinct eIF4E conformational changes. Additional interactions occur between Ascaris eIF4E and the SL on binding the m2,2,7G-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 m2,2,7G-cap is present. These data have implications for the contribution of 5′-UTRs in mRNA translation and the function of different eIF4E isoforms.

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.

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

ABSTRACT 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′ m7G-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 m7G-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.

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 β‐ and γ‐phosphate groups in the triphosphate chain. The kinetic parameters of enzymatic hydrolysis (Km, Vmax) 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 m32,2,7GpppG and m32,2,7GpppA 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 Km 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.

Affinity of dinucleotide cap analogues for human decapping scavenger (hDcpS).

Eukaryotic cells utilize scavenger decapping enzymes to degrade cap structure following 3′-5′ mRNA decay. Human DcpS recently has been described as a highly specific hydrolase (a member of the HIT family) that catalyses the cleavage of m 7 GpppG and short capped oligoribonucleotides. We have demonstrated here that cap-1 (m 7 GpppGm) is a preferred substrate among several investigated dinucleotide cap analogues m 7 Gp n N (n = 3–5, N is a purine or pyrimidine base) and m 7 GMP is always one of the reaction product. Cap analogues containing pyrimidine base instead of guanine or diphosphate chain are resistant to hydrolysis catalyzed by human scavenger. Contrary to the other enzymes of HIT family, hDcpS activity is not stimulated by Mg 2+.

How to find the optimal partner--studies of snurportin 1 interactions with U snRNA 5' TMG-cap analogues containing modified 2-amino group of 7-methylguanosine.

Snurportin 1 is an adaptor protein that mediates the active nuclear import of uridine-rich small nuclear RNAs (U snRNA) by the importin-β receptor pathway. Its cellular activity influences the overall transport yield of small ribonucleoprotein complexes containing N(2),N(2),7-trimethylguanosine (TMG) capped U snRNA. So far little is still known about structural requirements related to molecular recognition of the trimethylguanosine moiety by snurportin in solution. Since these interactions are of a great biomedical importance, we synthesized a series of new 7-methylguanosine cap analogues with extended substituents at the exocyclic 2-amino group to gain a deeper insight into how the TMG-cap is adapted into the snurportin cap-binding pocket. Prepared chemical tools were applied in binding assays using emission spectroscopy. Surprisingly, our results revealed strict selectivity of snurportin towards the TMG-cap structure that relied mainly on its structural stiffness and compactness.