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. 2005 Jan;11(1):77-89.
doi: 10.1261/rna.7108205.

High affinity RNA for mammalian initiation factor 4E interferes with mRNA-cap binding and inhibits translation

Kiyotaka Mochizuki  1 , Akihiro Oguro, Takashi Ohtsu, Nahum Sonenberg, Yoshikazu Nakamura
Affiliations

High affinity RNA for mammalian initiation factor 4E interferes with mRNA-cap binding and inhibits translation

Kiyotaka Mochizuki et al. RNA. 2005 Jan.

Abstract

The eukaryotic translation initiation factor 4F (eIF4F) consists of three polypeptides (eIF4A, eIF4G, and eIF4E) and is responsible for recruiting ribosomes to mRNA. eIF4E recognizes the mRNA 5'-cap structure (m7GpppN) and plays a pivotal role in control of translation initiation, which is the rate-limiting step in translation. Overexpression of eIF4E has a dramatic effect on cell growth and leads to oncogenic transformation. Therefore, an inhibitory agent to eIF4E, if any, might serve as a novel therapeutic against malignancies that are caused by aberrant translational control. Along these lines, we developed two RNA aptamers, aptamer 1 and aptamer 2, with high affinity for mammalian eIF4E by in vitro RNA selection-amplification. Aptamer 1 inhibits the cap binding to eIF4E more efficiently than the cap analog m7GpppN or aptamer 2. Consistently, aptamer 1 inhibits specifically cap-dependent in vitro translation while it does not inhibit cap-independent HCV IRES-directed translation initiation. The interaction between eIF4E and eIF4E-binding protein 1 (4E-BP1), however, was not inhibited by aptamer 1. Aptamer 1 is composed of 86 nucleotides, and the high affinity to eIF4E is affected by deletions at both termini. Moreover, relatively large areas in the aptamer 1 fold are protected by eIF4E as determined by ribonuclease footprinting. These findings indicate that aptamers can achieve high affinity to a specific target protein via global conformational recognition. The genetic mutation and affinity study of variant eIF4E proteins suggests that aptamer 1 binds to eIF4E adjacent to the entrance of the cap-binding slot and blocks the cap-binding pocket, thereby inhibiting translation initiation.

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Figures

FIGURE 1.
FIGURE 1.
In vitro selected RNA sequences and their affinities for eIF4E. (A) Representative RNA sequences selected from randomized N40 RNA libraries. The sequence of the parental N40 RNA pool contains 5′ and 3′ constant sequences for primer annealing. After 14 rounds of selection, 144 individual clones were selected and nine nonhomologous sequences were identified. The frequency of each sequence in these selections is shown as numbers of each clone found in 144 independent isolates. (B) Nitrocellulose filter binding assays of selected RNAs and N40 random RNA (control) for wild-type eIF4A. Shown is the percentage of input [32P]-labeled RNA bound to the nitrocellulose filter. RNAs no. 15 and no. 34 showed efficient binding activity to eIF4E and are referred to as aptamer 1 and aptamer 2, respectively. (C) Sensorgrams of eIF4E binding to N40 random RNA (left panel, control) and aptamer 1 (right panel). Each eIF4E sample at the indicated concentrations were injected to flow cells immobilized with either aptamer 1 or N40.
FIGURE 2.
FIGURE 2.
Inhibition of the m7GTP–eIF4E interaction by RNA aptamers. (A) Pull-down assay of an eIF4E and m7GTP–Sepharose binary complex. eIF4E and m7GTP–Sepharose were mixed and challenged by increasing amounts of the indicated competitors—free m7GTP cap analog, aptamer 1, and aptamer 2. eIF4E remaining bound to m7GTP–Sepharose was detected by Coomassie staining after SDS-PAGE. (B) The intensity of eIF4E bands associated with m7GTP–Sepharose was quantified using NIH Image J, and the data are plotted using Kaleida Graph software as a percentage of the bound eIF4E in the absence of competitor: m7GTP (circle), N40 (square), aptamer 1 (diamond), aptamer 2 (cross).
FIGURE 3.
FIGURE 3.
Identification of the aptamer · eIF4E · 4E-BP1 ternary complex by SPR analysis. The aptamer 1 sensor chip was injected with 100 nM eIF4E for 60 sec to a signal of 80 RUs and then challenged with the indicated amounts of (A) GST and (B) GST-4E-BP1 at time 0 for 60 sec. A series of sensorgrams is normalized to represent the net interaction between (A) RNA and GST or (B) RNA and GST–4E-BP1. Experimental conditions and procedures are described in Materials and Methods.
FIGURE 4.
FIGURE 4.
Inhibition of cap-dependent in vitro translation by aptamer 1. (A) Pull-down of RRL-bearing endogenous eIF4E with m7GTP–Sepharose in the presence of N40 random and aptamer 1 RNAs. RRL and m7GTP–Sepharose were mixed, and the indicated amounts of N40 random RNA (control) and aptamer 1 were introduced as competitors. Pulled-down eIF4E was detected by immunostaining after SDS-PAGE as described in Materials and Methods. (B) Schematic diagram of capped CAT/HCV-IRES/LUC mRNA. (C) Translation products of capped CAT/HCV-IRES/LUC mRNA in RRL. Reaction mixtures were preincubated at 30°C for 3 min with increasing amounts (0.5, 1.0, 2.5, 5.0 μM) of N40 and aptamer 1 RNAs, followed by the addition of mRNA and [35S] methionine and further incubation for 60 min at 30°C. To stimulate cap-dependent translation as well as to avoid any nonspecific (inhibitory) effect of N40 (control) RNA on the in vitro translation, the reaction mix (25 μL) contained an increased amount (17.5 μL) of RRL, 100 mM potassium acetate, and 0.5 mM magnesium acetate. Products were analyzed by SDS-PAGE (15%) and fluorography. [35S]methionine incorporated into CAT and LUC is quantified using BAS-2000 PhosphorImager (Fuji Co.), and their relative values (CAT/LUC) are shown. The CAT/LUC ratio obtained in the absence of RNAs (left lane, buffer control) was set as 100%.
FIGURE 5.
FIGURE 5.
The MFOLD prediction and structural probing and footprinting of aptamer 1 by RNase digestion. (A) The 5′-end 32P-labeled aptamer 1 was digested with RNase A, T1 (left), and V1 (right) in the presence or absence of eIF4E, and the resulting digests were separated by electrophoresis on urea-denaturing gels as described in Materials and Methods. Undigested aptamer 1 (1/10 volume) and alkaline-digested ladders are also run (lanes ap1 and –OH, respectively). Signals generated by nuclease cleavage are assigned with nucleotide positions on both sides of the lanes. (B) The secondary structure of aptamer 1 examined by ribonuclease sensitivity and eIF4E protection assays. Solid arrowheads indicate the cleavage points with RNase A and T1, and open arrowheads indicate RNase V1 cleavage positions. The arrowhead size represents the degree of cleavage. The bases enclosed in black squares indicate sites protected by the addition of eIF4E from RNase A, T1, and V1 hydrolysis.
FIGURE 6.
FIGURE 6.
Characterization of variant eIF4E proteins changed at affected in functional amino acids. A set of amino acid changes was generated in eIF4E by site-directed mutagenesis, and the resulting variant proteins were purified and examined as described in Materials and Methods. (A) The activity of variant eIF4Es for binding to the m7GTP cap analog and GST–4E-BP1 was examined by respective pull-down assays. The same amount of variant eIF4E proteins (top panel) was applied to each pull-down assay, and those coprecipitated with m7GTP–Sepharose (middle panel) and GST–4E-BP1 (bottom panel) were eluted and detected by Coomassie staining after SDS-PAGE. (B) The intensity of each band derived from both pull-downs was measured by NIH image J application and used to estimate the relative efficiency (compared to wild-type eIF4E) of binding to m7GTP–Sepharose (closed box) and GST–4E-BP1 (open box). In the GST–BP1 pull-down, the ratio of variant eIF4E to 4E-BP1 in the staining was compared with that of wild-type eIF4E to 4E-BP1 (to normalize the efficiency of GST-4E-BP1 precipitation). (C) Sensorgrams of the interaction between aptamer 1 and increasing amount of variant eIF4Es.
FIGURE 7.
FIGURE 7.
Structure of the mouse eIF4E (amino acids 28–217) and m7GTP complex (Protein Data Bank accession code 1L8B) (Niedzwiecka et al. 2002). Structure modeling is performed by Swiss PDB Viewer application and rendered by POV-Ray (ver. 3.6). (A) Mutation sites are shown on the ribbon model of the eIF4E–m7GTP complex structure. Amino acids altered in this study are indicated as space-filling presentations showing (green) stacking, (red) acidic, (blue) basic, and (orange) phosphorylation residues. m7GTP is displayed as color-coded space filling—(white) carbon, (red) oxygen, (sky blue) nitrogen, (yellow) phosphorus—and located in the cap-binding slot in the cocrystal. (B) Surface electrostatic potential of the eIF4E–m7GTP complex structure. (Blue) positive (basic amino acid) and (red) negative (acidic amino acid) charges are shown. Arg 112 and Lys 206 residues required for aptamer 1 binding are yellow. Arg 157 that is not involved in aptamer 1 binding is green. m7GTP is shown as in A. The potentials were calculated with the program DELPHI.
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