Structural characterization of eukaryotic mRNA decay factors involved in post-transcriptional gene regulation

Abstract

In animal cells, miRNAs silence the expression of mRNA targets through translational repression and/or deadenylation. Silencing requires the association of miRNAs to an Argonaute protein, which moreover binds to a member of the GW182/TNRC6C protein family. In turn, GW182 proteins interact with the two major cytoplasmic deadenylase complexes, the PAN2-PAN3 complex and the CCR4-NOT complex, to induce deadenylation and hence promote decay of miRNA targets. Structural insights into the assembly of the CCR4-NOT and PAN2-PAN3 complexes could be obtained by determining several sub-complexes of these multi-subunit deadenylases, including the crystal structures of the NOT1-CAF40 complex, the minimal NOT module (composed of the C-terminal domains of NOT1, NOT2 and NOT3) and the crystal structure of the folded domains of the PAN3 adaptor protein. Nevertheless, the most remarkable features were the identification of tryptophan-binding pockets in CAF40/CNOT9 and PAN3, providing a rational basis for the recruitment of the deadenylases to miRNA targets through GW182/TNRC6 proteins. Finally, the first crystal structure of the MID-PIWI lobe from a eukaryotic Argonaute protein was solved, which provided first molecular insights into eukaryotic Argonaute biology.