Structural Insights into Parasite eIF4E Binding Specificity for m7G and m2,2,7G mRNA Caps

Abstract

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 eIF4E⅐m 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 capbinding 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. Eukaryotic initiation protein eIF4E2 is an essential translation factor that recognizes the mRNA cap (1-3). Recognition of the mRNA cap by eIF4E is the key and rate-limiting step in mRNA translation. The majority of translation in eukaryotic cells is cap-dependent; that is recruitment of mRNAs to the ribosome for translation is dependent on the interaction between eIF4E and the mRNA cap. eIF4E directly binds to the mRNA cap. However, for productive translation initiation to occur, eIF4E must interact with eIF4G. eIF4G acts as a bridge protein interacting with factors in the 40 S ribosomal subunit that facilitate ribosome recruitment to the mRNA. Increased expression of eIF4E is associated with a variety of cancers and cancer progression (4). Efforts in a number of laboratories are directed toward therapies against eIF4E in cancer, including the development of cap analogs (5-9).The mRNA cap in most eukaryotes is m 7 GpppN (where N is A, C, G, or U). The cap contains a 5Ј-5Ј triphosphate bridge with the first guanosine methylated at the N-7 position. However, spliced leader trans-splicing in metazoa adds a different cap to recipient mRNAs, a trimethylguanosine cap, m 2,2,7 GpppN (see Fig. 1A) (10 -14). trans-splicing is present in a variety of parasitic nematodes and flatworms, and these organisms remain a significant health problem in many parts of the world, infecting upward of 2 billion people (15-17). Translation of these trans-spliced mRNAs is thought to require eIF4E recognition of the m 2,2,7 G cap to facilitate ribosomal ...

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“…The large number of residues that undergo CSPs is in agreement with the enclosure of the cap structure into the core of the eIF4E protein (Matsuo et al 1997;Kinkelin et al 2012). In agreement with the limited (Tomoo et al 2005;Liu et al 2009;Peter et al 2015) show that the position of the first nucleotide of the RNA body (pink) is not well defined. This indicates that the interaction between eIF4e and the mRNA is mediated almost exclusively through the m 7 GTP part of the cap-0 structure.…”

Recent advances in Echinococcus genomics and stem cell research

“…The development of respective, parasite-specific drugs could prove difficult if they are directed against the splicing process itself, since trans-splicing is carried out by the canonical spliceosome, the components of which are highly conserved between cestodes and mammals (Tsai et al, 2013). However, as already suggested previously (Liu et al, 2009), the eukaryotic translation initiation factor 4E (eIF4E), which initiates translation by binding to the mRNA cap structure, could be a promising target. In mammals, eIF4E only recognises 7mG cap structures, whereas in the trans-splicing schistosomes an eIF4E is present that recognises both 7mG and TMG caps (Liu et al, 2009).…”

“…However, as already suggested previously (Liu et al, 2009), the eukaryotic translation initiation factor 4E (eIF4E), which initiates translation by binding to the mRNA cap structure, could be a promising target. In mammals, eIF4E only recognises 7mG cap structures, whereas in the trans-splicing schistosomes an eIF4E is present that recognises both 7mG and TMG caps (Liu et al, 2009). The E. multilocularis genome contains one single copy gene for eIF4E and it is reasonable to assume that this factor also recognises both types of caps.…”

“…One striking difference in gene expression mechanisms between flatworm parasites and their hosts, which has already been suggested to be exploitable for drug development (Liu et al, 2009), is spliced-leader trans-splicing (SL-TS). Originally described in trypanosomes and nematodes (Hastings, 2005), SL-TS has later also been shown to occur in all parasitic and free-living lineages of flatworms (Brehm et al, 2002(Brehm et al, , 2000aDavis et al, 1995;Zayas et al, 2005).…”

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“…[37][38][39]. Total lysates, washed beads after m 7 GTP co-capture, and the supernatants were Western-blotted for eIF4E, 4E-BP, or eIF4G.…”