Wheat Eukaryotic Initiation Factor 4B Organizes Assembly of RNA and eIFiso4G, eIF4A, and Poly(A)-binding Protein

Shijun Cheng,Daniel R Gallie

doi:10.1074/jbc.m605404200

Shijun Cheng, Daniel R Gallie

Open Access

https://doi.org/10.1074/jbc.m605404200

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Abstract

The eukaryotic translation initiation factor (eIF) 4B promotes the RNA-dependent ATP hydrolysis activity and ATP-dependent RNA helicase activity of eIF4A and eIF4F during translation initiation. Although this function is conserved among plants, animals, and yeast, eIF4B is one of the least conserved of initiation factors at the sequence level. To gain insight into its functional conservation, the organization of the functional domains of eIF4B from wheat has been investigated. Plant eIF4B contains three RNA binding domains, one more than reported for mammalian or yeast eIF4B, and each domain exhibits a preference for purine-rich RNA. In addition to a conserved RNA recognition motif and a C-terminal RNA binding domain, wheat eIF4B contains a novel N-terminal RNA binding domain that requires a short, lysine-rich containing sequence. Both the lysine-rich motif and an adjacent, C-proximal motif are conserved with an N-proximal sequence in human and yeast eIF4B. The C-proximal motif within the N-terminal RNA binding domain in wheat eIF4B is required for interaction with eIFiso4G, an interaction not reported for other eIF4B proteins. Moreover, each RNA binding domain requires dimerization for binding activity. Two binding sites for the poly(A)-binding protein were mapped to a region within each of two conserved 41-amino acid repeat domains on either side of the C-terminal RNA binding domain. eIF4A bound to an adjacent region within each repeat, supporting a central role for these conserved eIF4B domains in facilitating interaction with other components of the translational machinery. These results support the notion that eIF4B functions by organizing multiple components of the translation initiation machinery and RNA.

Highlights

EIF4F, recruits several other factors, including eIF4A, eIF3, and the poly(A)-binding protein (PABP) through direct proteinprotein interactions (1– 6)
We show here that wheat eIF4B contains three RNA binding domains, two binding sites for eIF4A and PABP, and one binding site for eIFiso4G (Fig. 10) that provides for the first time a physical basis for the known functional interactions between eIF4B and other components of the translational machinery
The organization of the RNA binding domains in wheat eIF4B is similar to mammalian eIF4B with one corresponding to the RNA recognition motif (RRM) and another in the central region of the protein

Summary

Introduction

EIF4F, recruits several other factors, including eIF4A, eIF3, and the poly(A)-binding protein (PABP) through direct proteinprotein interactions (1– 6). Truncation to residue 310 (i.e. GST-eIF4B-(69 –310)) exhibited just detectable RNA binding activity, whereas further deletion to amino acid 300 (i.e. GST-eIF4B-(69 –300)) abolished RNA binding (Fig. 2C, lanes 3 and 4, respectively, bottom panel).

Results

Conclusion