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

Nicholas M Kaye,Kelly J Emmett,William C Merrick,Eckhard Jankowsky

doi:10.1074/jbc.m109.009001

Nicholas M Kaye, Kelly J Emmett + Show 2 more

Open Access

https://doi.org/10.1074/jbc.m109.009001

Copy DOI

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.

Highlights

The first translation initiation factor to bind the mRNA during cap-dependent eukaryotic translation initiation is thought to be the eukaryotic initiation factor 4F [1,2,3,4]. eIF4F is a stable tripartite protein complex composed of the scaffolding and RNA-binding protein eIF4G, the DEAD-box protein eIF4A, and the m7G cap-binding protein eIF4E [1]. eIF4E is the only known initiation factor to directly contact the m7G cap [1]
By measuring equilibrium binding of rabbit eIF4F to a diverse series of capped and uncapped RNAs, we found no significant effect of the cap on the stability of eIF4F-RNA complexes, even though the m7G cap was verified to be essential for efficient translation initiation
These observations show that intrinsic RNA binding by eIF4F depends on RNA length, not on m7G cap recognition, and these binding characteristics are recapitulated in rabbit reticulocyte lysate (RRL)

Summary

EXPERIMENTAL PROCEDURES

Protein Purification— eIF4F and all other proteins used in this study were purified from RRL and quantified as described previously [11,12,13,14]. RNA Preparation—RNAs were either generated by in vitro transcription or purchased from Dharmacon The latter were deprotected, purified, and quantified as described [15, 16]. EIF4F-RNA Binding Reactions— eIF4F-RNA binding was measured at the eIF4F concentrations indicated at 37 °C in a reaction volume of 10 ␮l with buffer containing 2.5 mM MgCl2, 100 mM KCl, 0.1 mM EDTA, 8% (v/v) glycerol, 40 mM HEPES, pH 7.2, 0.3 mM dithiothreitol, and 0.01% Nonidet P-40 (v/v). Bound and free RNAs were separated by nondenaturing PAGE, as described above, except that glycerol was omitted from the gel. Reactions were performed in a volume of 7.5 ␮l, with 60% (v/v) RRL, 20 ng/␮l Promega luciferase control RNA (RNA concentrations were saturating; data not shown), and inhibitor RNAs, as indicated, for 30 min at 30 °C (linear phase of translation).

RESULTS

Findings

DISCUSSION