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Abstract:
The shortening of poly(A)-tails of mature mRNAs influences their translation status and is usually the first and rate- limiting step in eukaryotic mRNA turnover. Deadenylation is thought to occur in two phases, of which the initial one is catalyzed by the PAN2-PAN3 complex. Despite its important function and high conservation, structural information of this complex was so far limited to isolated PAN3. PAN2 is a multidomain protein comprising an N-terminal WD40 domain, a central ubiquitin specific protease domain, and a C-terminal 3′-5′ exoribonuclease domain of the DEDD family. The globular domains of PAN2 are connected by two low complexity regions. Although it is clear that the nuclease domain of the protein is responsible for PAN2 deadenylase activity, little is known about the role of the WD40 and USP domains in PAN2 function. Furthermore the molecular basis for the assembly of the PAN2-PAN3 complex remained unclear. To address these open questions, we solved crystal structures of all three folded domains of PAN2, i.e. the WD40, protease and exonuclease domains, as well as a structure of the PAN2-PAN3 complex. The structures reveal that the PAN2 exonuclease domain forms a tight assembly with the protease domain. In this arrangement, canonical exonuclease elements are replaced by parts of the protease domain, suggesting that the two domains coevolved to function as one unit. In contrast to previous studies, we show that complex formation with the PAN3 homodimer is mediated by the N-terminal WD40 domain of PAN2 together with the subsequent linker region. Surprisingly, our biochemical and structural data suggest that the asymmetry of the PAN3 homodimer together with the placement of the PAN2 linker prevent the recruitment of a second copy of PAN2. Thereby a molecular ratio of 1:2 is established in the fully assembled PAN2-PAN3 complex, that could have mechanistic implications.
