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. 2009 Jun 26;284(26):17742-50.
doi: 10.1074/jbc.M109.009001. Epub 2009 May 4.

Intrinsic RNA Binding by the Eukaryotic Initiation Factor 4F Depends on a Minimal RNA Length but Not on the m7G Cap

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Intrinsic RNA Binding by the Eukaryotic Initiation Factor 4F Depends on a Minimal RNA Length but Not on the m7G Cap

Nicholas M Kaye et al. J Biol Chem. .
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Abstract

The eukaryotic initiation factor 4F (eIF4F) is thought to be the first factor to bind mRNA during 7-methylguanosine (m7G) cap-dependent translation initiation. The multipartite eIF4F contains the cap-binding protein eIF4E, and it is assumed that eIF4F binds mRNAs primarily at the 5' m7G cap structure. We have analyzed equilibrium binding of rabbit eIF4F to a series of diverse RNAs and found no impact of the 5'-cap on the stability of eIF4F-RNA complexes. However, eIF4F preferentially and cooperatively binds to RNAs with a minimum length of approximately 60 nucleotides in vitro. Furthermore, translation activity in rabbit reticulocyte lysate is strongly inhibited by RNAs exceeding this length, but not by shorter ones, consistent with the notion that eIF4F in its physiological environment preferentially binds longer RNAs, too. Collectively, our results indicate that intrinsic RNA binding by eIF4F depends on a minimal RNA length, rather than on cap recognition. The nonetheless essential m7G cap may either function at steps subsequent to eIF4F-RNA binding, or other factors facilitate preferential binding of eIF4F to the m7G cap.

Figures

FIGURE 1.
FIGURE 1.
eIF4F binding to capped and uncapped RNA. A, representative nondenaturing PAGE of eIF4F (0, 5, 8, 15, 20, 25, 30, 50, and 90 nm) binding to radiolabeled uncapped (top) and capped (bottom) 158-nt RNA (1 nm). Mobilities of free and bound RNA are indicated on the left. B, representative plot of the dependence of the fraction of bound RNA on eIF4F concentration. Lines represent the best fit to the nonlinear form of the Hill equation (“Experimental Procedures”).
FIGURE 2.
FIGURE 2.
Toeprint analysis of capped and uncapped RNA. The sequence ladder indicates the AUG start codon. Primer extension of the β-globin RNA (full extension) and toeprint are indicated on the right. Presence (+) or absence (−) of an m7G cap on the RNA is indicated at the top of the gel. Lanes 3 and 4 (80S) denote reactions in the presence of cycloheximide; lanes 5 and 6 (48S) denote reactions in the presence of GMP-PNP. The fraction toeprint, indicated in the bar graph, represents the fraction of cpm at +14 to +18 compared with cpm for full extension. The values shown are the average, and the error bars indicate the standard deviation of multiple independent experiments.
FIGURE 3.
FIGURE 3.
Native Western blot for eIF4E before and after eIF4F-RNA binding. Western blot of eIF4F in the presence and absence of RNA on nondenaturing PAGE probed with anti-eIF4E. To generate free eIF4E (95 °C), 350 nm eIF4F was incubated with SDS at 95 °C for 2 min.
FIGURE 4.
FIGURE 4.
Experimental design probing the minimal RNA-binding site of eIF4F. Step 1, labeled (asterisk) capped or uncapped RNA is subjected to limited alkaline hydrolysis. The extent of hydrolysis is monitored by denaturing PAGE. Step 2, pool of hydrolyzed RNA is used as a substrate for eIF4F binding. The free and eIF4F-bound RNAs are excised and eluted from nondenaturing PAGE. Step 3, eIF4F-bound and free RNAs are purified and separated by denaturing PAGE.
FIGURE 5.
FIGURE 5.
Determination of the minimal binding site size of eIF4F. A and B, representative PAGE of eIF4F bound (B) and free (F) uncapped (A) and capped (B) RNAs (i.e. step 3 in Fig. 4) at increasing eIF4F levels. Concentrations of eIF4F are marked at top of panels. Size markers (M) are shown at the left of panels. Plot range indicates the individual RNA lengths shown in the quantitative analysis below. C and D, plots of normalized band intensities for bound fractions of uncapped (C) and capped (D) RNAs around the inflection point between bound and free RNA. Values represent averages of three independent measurements, and the error bars indicate the standard deviation. Band intensities for the independent experiments were normalized to bound and free RNA species, to facilitate comparison between the measurements. The smaller errors for the reaction with capped RNA reflect the higher signal of the radiolabeled RNAs.
FIGURE 6.
FIGURE 6.
Translation inhibition by different RNAs in rabbit reticulocyte lysate. A, inhibition by RNAs and DNAs of different lengths. fLuc synthesized indicates measured light units with inhibitor normalized to the light units without inhibitor, given in percent. Total nucleotide represents the number of nucleotides added with the RNAs of different length, as explained in the text. Error bars represent the standard deviation of at least three independent measurements. Sequences of the inhibitors are given in Table 1. B, inhibition by poly(IC). Total nucleotide represents the number of nucleotides added, as determined from absorption measurements at 260 nm. Inhibition was measured with an RRL lot different from the one used in the measurements shown in A. For normalization, an inhibition curve with RNA 66 is shown for this lot.
FIGURE 7.
FIGURE 7.
RNA length dependence of translation inhibition. Inhibition is given at 500 μm total nucleotide. The degree of inhibition is measured as in Fig. 6. Values represent averages of at least three independent measurements, and the error bars indicate the standard deviation. Inhibitor length is represented visually and by actual nucleotide number. Capped RNAs as well as DNA oligonucleotides are labeled accordingly. RNA and DNA sequences are given in Table 1.

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