Thermodynamics of mRNA 5‘ Cap Binding by Eukaryotic Translation Initiation Factor eIF4E

Niedzwiecka, Anna; Darżynkiewicz, Edward; Stolarski, Ryszard

doi:10.1021/bi0491651

Cited by 41 publications

(61 citation statements)

References 74 publications

(122 reference statements)

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“…This model suggests that intrinsic and specific conformational flexibility of the schistosome eIF4E plays the crucial role for m 2,2,7 G cap binding. Steric hindrance of the cap due to the additional two methyl groups at the N 2 position of the m 2,2,7 G cap has been suggested previously to likely be important in the reduced affinity of mammalian eIF4E in binding m 2,2,7 G cap (9,21). Previous mutagenesis studies for C. elegans eIF4E-5, a form of eIF4E that can bind both types of caps, showed that two residue changes (N64Y/V65L) (Fig.…”

Section: Discussionmentioning

confidence: 75%

“…Fluorescence intensity was corrected for the inner filter effect, and the emission of the free cap analogs was explicitly included in the numerical analysis. A theoretical curve for the fluorescence intensity as a function of the total ligand concentration was fitted to the experimental data points by means of non-linear, least squares method using Prism 3.02 (GraphPad Software) as described previously (9,30,41).…”

Section: Methodsmentioning

confidence: 99%

“…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)(6)(7)(8)(9).…”

Section: Eukaryotic Initiation Protein Eif4ementioning

confidence: 99%

“…Translation of these trans-spliced mRNAs is thought to require eIF4E recognition of the m 2,2,7 G cap to facilitate ribosomal recruitment (18 -20). Vertebrate eIF4E has very low affinity for the trimethylguanosine cap in comparison with the monomethylguanosine cap, and association of the m 2,2,7 G cap with mammalian eIF4E seems to destabilize the overall structure of the protein (1,9,21). Approximately 70% of mRNAs in the nematode Caenorhabditis elegans undergo trans-splicing.…”

Section: Eukaryotic Initiation Protein Eif4ementioning

confidence: 99%

“…Binding-The addition of a second base to an N-7 methylated nucleotide in general does not significantly increase mammalian eIF4E affinity for the cap (9). In fact, human eIF4E binding affinity for dinucleotide triphosphate cap (m 7 GpppG) is significantly lower than that observed for a mononucleotide (m 7 GTP) (9, 45) Interestingly, the schistosome eIF4E binding affinity for mono-and dinucleotide substrates is similar as determined by ITC (Table 2 and data not shown).…”

Section: Other Insights From the Schistosome Eif4e Structure On Capmentioning

confidence: 99%

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

Liu

Zhao

McFarland

et al. 2009

Journal of Biological Chemistry

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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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Section: Discussionmentioning

confidence: 75%

Section: Methodsmentioning

confidence: 99%

Section: Eukaryotic Initiation Protein Eif4ementioning

confidence: 99%

Section: Eukaryotic Initiation Protein Eif4ementioning

confidence: 99%

Section: Other Insights From the Schistosome Eif4e Structure On Capmentioning

confidence: 99%

See 3 more Smart Citations

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

Liu

Zhao

McFarland

et al. 2009

Journal of Biological Chemistry

Self Cite

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Applications of isothermal titration calorimetry in RNA biochemistry and biophysics

Feig

2007

Biopolymers

100

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Isothermal titration calorimetry (ITC) has been applied to the study of proteins for many years. Its use in the biophysical analysis of RNAs has lagged significantly behind its use in protein biochemistry, however, in part because of the relatively large samples required. As the instrumentation has become more sensitive, the ability to obtain high quality data on RNA folding and RNA ligand interactions has improved dramatically. This review provides an overview of the ITC experiment and describes recent work on RNA systems that have taken advantage of its versatility for the study of small molecule binding, protein binding, and the analysis of RNA folding. © 2007 Wiley Periodicals, Inc. Biopolymers 87: 293–301, 2007. This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com

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Fluorescent Turn‐On Probes for the Development of Binding and Hydrolytic Activity Assays for mRNA Cap‐Recognizing Proteins

Kasprzyk

Starek

Ciechanowicz

et al. 2019

Chemistry A European J

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The m 7 Gc ap is au nique nucleotide structure at the 5'-end of all eukaryotic mRNAs. The cap specificallyinteracts with numerous cellular proteins and participates in biological processes that are essential for cell growth and function. To provide small molecularp robest os tudy important cap-recognizing proteins, we synthesized m 7 Gn ucleotides labeled with fluorescent tags via the terminal phosph(on)ate group and studied how their emission properties changed upon protein binding or enzymaticc leavage. Only the pyrene-labeled compounds behaved as sensitive turn-on probes. Ap yrene-labeled m 7 GTP analogue showed up to eightfold enhanced fluorescencee mission upon binding to eukaryotic translation initiation factor 4E (eIF4E) and over 30-folde nhancementu pon cleavageb yd ecapping scavenger (DcpS) enzyme. These observations serveda st he basis for developing binding-and hydrolytic-activity assays. The assay utility was validated with previously characterizedl ibraries of eIF4E ligands and DcpS inhibitors. The DcpS assay was also appliedtostudy hydrolytic activity and inhibition of endogenous enzyme in cytoplasmic extracts from HeLa and HEK cells.

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Thermodynamics of mRNA 5‘ Cap Binding by Eukaryotic Translation Initiation Factor eIF4E

Cited by 41 publications

References 74 publications

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

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

Applications of isothermal titration calorimetry in RNA biochemistry and biophysics

Fluorescent Turn‐On Probes for the Development of Binding and Hydrolytic Activity Assays for mRNA Cap‐Recognizing Proteins

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