Overlapping regions of Caf20 mediate its interactions with the mRNA-5\u2032cap-binding protein eIF4E and with ribosomes

Ebelechukwu C Nwokoye,Graham D Pavitt,Martin D Jennings,Eiman Alnaseem,Robert A Crawford,Christopher J Kershaw,Lydia M Castelli

doi:10.1038/s41598-021-92931-4

Abstract

By interacting with the mRNA 5′ cap, the translation initiation factor eIF4E plays a critical role in selecting mRNAs for protein synthesis in eukaryotic cells. Caf20 is a member of the family of proteins found across eukaryotes termed 4E-BPs, which compete with eIF4G for interaction with eIF4E. Caf20 independently interacts with ribosomes. Thus, Caf20 modulates the mRNA selection process via poorly understood mechanisms. Here we performed unbiased mutagenesis across Caf20 to characterise which regions of Caf20 are important for interaction with eIF4E and with ribosomes. Caf20 binding to eIF4E is entirely dependent on a canonical motif shared with other 4E-BPs. However, binding to ribosomes is weakened by mutations throughout the protein, suggesting an extended binding interface that partially overlaps with the eIF4E-interaction region. By using chemical crosslinking, we identify a potential ribosome interaction region on the ribosome surface that spans both small and large subunits and is close to a known interaction site of eIF3. The function of ribosome binding by Caf20 remains unclear.

Highlights

By interacting with the mRNA 5′ cap, the translation initiation factor eIF4E plays a critical role in selecting mRNAs for protein synthesis in eukaryotic cells
We extended this analysis in two ways, first by independently deleting either the canonical or the NC helices to create the C af20∆1 and C af20∆2 mutants, each removing 20 amino acids from the full-length protein (Fig. 1b) and evaluating their binding to eIF4E
Caf[20] shares the canonical 4E-BP motif, but unlike other 4E-BPs this motif is found directly at its amino terminus (Fig. S2). It was proposed following in vitro binding studies with a Caf[20] N-terminal peptide that unlike other 4E-BPs, the NC helix does not contribute to the strength of the eIF4E interaction, despite forming part of the interface[9]

Summary

Introduction

By interacting with the mRNA 5′ cap, the translation initiation factor eIF4E plays a critical role in selecting mRNAs for protein synthesis in eukaryotic cells. Caf[20] binding to eIF4E is entirely dependent on a canonical motif shared with other 4E-BPs. binding to ribosomes is weakened by mutations throughout the protein, suggesting an extended binding interface that partially overlaps with the eIF4E-interaction region. Altmann and colleagues found that Caf[20] could enhance translation in in vitro translation extracts programmed with specific m RNAs16 These observations suggest that Caf[20] may function with binding partners in addition to eIF4E and have a more complex role in translation. We find and evaluate three potential ribosomal protein partners found on one side of the ribosome away from the main tRNA and translation factor binding sites

Methods

Results

Conclusion