eIF4A2 drives repression of translation at initiation by Ccr4-Not through purine-rich motifs in the 5'UTR

Background: Regulation of the mRNA life cycle is central to gene expression control and determination of cell fate. miRNAs represent a critical mRNA regulatory mechanism, but despite decades of research, their mode of action is still not fully understood. Results: Here, we show that eIF4A2 is a major effector of the repressive miRNA pathway functioning via the Ccr4-Not complex. We demonstrate that while DDX6 interacts with Ccr4-Not, its effects in the mechanism are not as pronounced. Through its interaction with the Ccr4- Not complex, eIF4A2 represses mRNAs at translation initiation. We show evidence that native eIF4A2 has similar RNA-selectivity to chemically inhibited eIF4A1. eIF4A2 exerts its repressive effect by binding purine-rich motifs which are enriched in the 5'UTR of target mRNAs directly upstream of the AUG start codon. Conclusions: Our data support a model whereby purine motifs towards the 3' end of the 5'UTR are associated with increased ribosome occupancy and possible uORF activation upon eIF4A2 binding. Overall design: RIP-Seq of endogenous IPs of eIF4A1, eIF4A2 ad DDX6 with total RNA; RNA-Seq of subpolysomal and polysomal fractions from sucrose density gradients from HEK293 cells treated with control siRNA or CNOT1 siRNA with total RNA; RNA-Seq of total RNA of HEK293 cells transfected with either a control siRNA or siRNA against TNRC6A and TNRC6B