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Abstract

The PAN2-PAN3 deadenylase complex functions in general and miRNA-mediated mRNA degradation and is specifically recruited to miRNA targets by the TNRC6 proteins. PAN3 is an adaptor protein that recruits the PAN2 deadenylase to mRNA targets. PAN3 contains three prominent regions: an N-terminal region that is predicted to be unstructured, a central pseudokinase (PK) domain and a highly conserved C-terminal domain (C-term), which is unique to the PAN3 protein family. While the N-terminal region of PAN3 contains a well-characterized PAM2 motif, studies on the roles of the PK and C-term domains have been restricted to the yeast protein. In particular, yeast two- hybrid assays have demonstrated that a C-term domain of yeast PAN3 is required for PAN2 binding, while both the PK+C-term domains mediated self-interaction. However, the stoichiometry and significance of PAN3 oligomerization has remained unclear. Here we show that PAN3 forms dimers in vitro and in vivo. Surprisingly, despite containing nonconservative substitutions in all the sequence motifs required for kinase activity, the PAN3 pseudokinase domain is capable of binding ATP and the integrity of the nucleotide binding pocket is required for deadenylation in vivo. Through a combined mutational and functional analysis, we have identified critical residues that mediate PAN3 interaction with PAN2 and TNRC6 proteins, as well as additional residues required for deadenylation in vivo. Collectively, our data provides a framework for understanding the role of PAN3 in recruiting PAN2 to mRNA targets to initiate their deadenylation and reveals a role for ATP-binding in mRNA deadenylation.