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

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.

Translation factor and RNA binding protein mRNA interactomes support broader RNA regulons for posttranscriptional control

“…2 A ) is that the eIF4A profile is highly similar to the profiles of the two yeast eIF4E binding proteins (4E-BPs), Eap1p and Caf20p (R 2 values of 0.73 and 0.65, respectively). Eap1p and Caf20p have been thought of as translation repressors acting to curtail translation initiation through interactions with eIF4E and/or via interactions with the ribosome ( 48 , 49 , 50 , 51 ). Indeed, both proteins interact with eIF4E to inhibit translation in vitro ( 52 , 53 ), and deletion of the CAF20 gene suppresses, whereas CAF20 overexpression exacerbates certain translation factor mutations, including mutations in eIF4A ( tif1-1 ) ( 54 ).…”

Yeast Tor complex 1 phosphorylates eIF4E‐binding protein, Caf20

“…Interaction of Caf20 with ribosomes and the 3′‐UTR region of mRNA in an eIF4E‐independent manner is also proposed (Castelli et al, 2015; Nwokoye et al, 2021). We generated an eIF4E‐binding motif mutant of Caf20 (Caf20‐BM), which loses affinity to eIF4E.…”

Nonviral Delivery Systems of mRNA Vaccines for Cancer Gene Therapy

“…There are three major types of 5′-cap structures, including type O, type I, and type II, which are classified according to whether there is a methylated ribose on the 2′ hydroxyl group of the first or second nucleotide from the 5′ end [ 43 ] ( Figure 4 ). The 5′-cap structures not only prevent IVT mRNA from being degraded by enzymes, such as alkaline phosphatase (AKP) and 5′ to 3′ exonuclease [ 44 ], but also can promote protein biosynthesis by forming starting complexes with the translation initiation factors eIF4E and eIF4G [ 45 , 46 ]. Moreover, the direction of the 5′-cap is very important, as indicated by a previous study demonstrating that mRNA with an inverted cap shows profoundly decreased translation efficiency [ 47 ].…”