Translational regulation impacts metazoan development, learning and human disease1–3
. Of the many RNA-binding proteins regulating mRNA stability, localization and translation, the two classes relevant to this work are the PUF and Ago families. PUF proteins regulate a broad spectrum of mRNAs to maintain stem cells, among other functions1,2
. Ago proteins bind small RNAs, most notably miRNAs, and act in many biological contexts, including stem cells (reviewed in 3).
PUF proteins regulate mRNA expression in virtually all eukaryotes4
. One conserved mechanism, which has been established for Saccharomyces cerevisiae
, Caenorhabditis elegans
and human PUF proteins, relies on the direct recruitment of the Ccr4-Not deadenylase complex to target mRNAs, resulting in a shorter poly(A)-tail length and either mRNA instability or translational repression5,6
. PUF proteins in yeast and flies also use deadenylation-independent mechanisms, although the specifics of these mechanisms remain largely unknown7,8
Ago proteins function within the miRISC complex to control target mRNA stability and translation. This complex controls deadenylation and stability of its target mRNAs9–11
, but its mechanism to control translation has garnered controversy. Best documented is its regulation of translation initiation9–13
. However, a mechanism affecting translation elongation also appears to exist since target mRNAs can be bound to polyribosomes14
. One model is that miRISC promotes ribosomal drop-off during translation elongation15
. Yet the specific mechanism by which miRISC, and hence Ago family members, inhibits translation elongation is not understood.
Here, we report that PUF and Ago proteins form an inhibitory complex with eEF1A, a GTPase required for translation elongation. FBF-1 (a C. elegans PUF protein) binds CSR-1 (a C. elegans Ago family member) in vitro and in vivo, and csr-1 depletion leads to increased expression of FBF target mRNAs. The FBF-1/CSR-1 heterodimer forms a complex with EFT-3 (C. elegans eEF1A), and this FBF-1/CSR-1/EFT-3 ternary complex has inhibited GTPase activity. Importantly, the PUF/Ago/eEF1A complex is conserved: human PUM2 (a PUF protein) associates with human AGO proteins in vivo and with eEF1A. Wild-type human PUM2 inhibits translation of both nonadenylated and polyadenylated mRNAs in rabbit reticulocyte lysate; however, PUM2 mutants that cannot form the PUM2/Ago/eEF1A complex or that cannot bind RNA are severely compromised for translation repression. Mechanistically, PUM2/Ago/eEF1A represses translation during elongation with ribosomes accumulating ~100–140 nts after the AUG within the open-reading frame (ORF). We propose a model in which PUF and AGO proteins complex with eEF1A to inhibit its GTPase activity and attenuate translation elongation.