Structural basis for Pan3 binding to Pan2 and its function in mRNA recruitment and deadenylation

Abstract

The conserved eukaryotic Pan2–Pan3 deadenylation complex shortens cytoplasmic mRNA 3′ polyA tails to regulate mRNA stability. Although the exonuclease activity resides in Pan2, efficient deadenylation requires Pan3. The mechanistic role of Pan3 is unclear. Here, we show that Pan3 binds RNA directly both through its pseudokinase/C‐terminal domain and via an N‐terminal zinc finger that binds polyA RNA specifically. In contrast, isolated Pan2 is unable to bind RNA. Pan3 binds to the region of Pan2 that links its N‐terminal WD40 domain to the C‐terminal part that contains the exonuclease, with a 2:1 stoichiometry. The crystal structure of the Pan2 linker region bound to a Pan3 homodimer shows how the unusual structural asymmetry of the Pan3 dimer is used to form an extensive high‐affinity interaction. This binding allows Pan3 to supply Pan2 with substrate polyA RNA, facilitating efficient mRNA deadenylation by the intact Pan2–Pan3 complex.

Towards a molecular understanding of microRNA-mediated gene silencing

“…In addition, PAN3 binds to RNA through an N-terminal zinc-finger domain 65 . The structure of PAN3 bound to a W-containing peptide (derived from a neighbouring PAN3 molecule in the crystal lattice), together with structures of PAN3 bound to PAN2 (REFS 28,65-67), provides a detailed molecular model for the assembly of the PAN2-PAN3 complex and its recruitment to miRNA targets.…”

The Functions of MicroRNAs: mRNA Decay and Translational Repression

“…The crystal structure of PAN3 forms intertwined and asymmetric homodimers [43,[140][141][142]. The crystal structure of the PAN2-PAN3 complex reveals that the catalytic subunit PAN2 and PAN3 interact with 1:2 stoichiometry [43,[140][141][142].…”

“…The crystal structure of the PAN2-PAN3 complex reveals that the catalytic subunit PAN2 and PAN3 interact with 1:2 stoichiometry [43,[140][141][142]. The PAN3 dimerization interface harbors a W-binding pocket, which is required for the interaction with TNRC6 [43].…”

“…This CNOT1-CNOT9 complex binds to TNRC6 through interaction between W motifs in TNRC6 and the two hydrophobic tryptophan-binding pockets located in the ARM repeat domain of the CNOT9 [45,46]. In addition to the CNOT9-mediated interaction, CNOT1 can interact with TNRC6 via the C-terminus region and the tristetraprolin-binding site of CNOT1 [45,46].The crystal structure of PAN3 forms intertwined and asymmetric homodimers [43,[140][141][142]. The crystal structure of the PAN2-PAN3 complex reveals that the catalytic subunit PAN2 and PAN3 interact with 1:2 stoichiometry [43,[140][141][142].…”

Regulatory roles of vertebrate Nocturnin: insights and remaining mysteries

“…5) [13,88]. Moreover, the discrepancy between NOCT in vivo and in vitro activity suggests that one or more protein partners is required for NOCT function; such a requirement has been observed previously for the PAN2 deadenylase [89,90]. Thus far, NOCT-interacting partners have not been comprehensively characterized, although one report using co-immunoprecipitation experiments in mammalian cells overexpressing tagged proteins demonstrated a physical interaction between mouse NOCT and PPARγ1 and PPARγ2 [18].…”