Hu proteins are RNA-binding proteins that stabilize adenine/uridine-rich element (ARE)-containing transcripts. We have recently shown that HuD, a member of the neuronal Hu family of proteins, stimulates translation of capped and polyadenylated mRNA. This stimulatory effect of HuD on translation depends on the interaction of HuD with eIF4A and a poly(A) tail. Thus, HuD is a powerful tool to positively modulate translation initiation via eIF4A.

Here, we applied biochemical approaches to directly examine the presence of key initiation factors on target mRNAs under repression. Using HEK293F cell extracts, we show that both eIF4AI and eIF4AII, but not other eIF4F components, dissociate from the mRNA in a miRISC-dependent manner at early time points. Conversely, HuD prevents the miRISC-induced dissociation of eIF4AI and eIF4AII via a direct interaction with the eIF4As, thereby relieving translational repression by miRISC. Furthermore, pharmacological treatment that prevents dissociation of eIF4As from mRNA conferred resistance to miRNA-mediated translation repression in vitro and in cultured cells. Our results provide a mechanistic explanation of how miRNAs repress the initiation step of translation.