Biological role of the two overlapping poly(A)-binding protein interacting motifs 2 (PAM2) of eukaryotic releasing factor eRF3 in mRNA decay

Abstract

Eukaryotic releasing factor GSPT/eRF3 mediates translation termination-coupled mRNA decay via interaction with a cytosolic poly(A)-binding protein (PABPC1). A region of eRF3 containing two overlapping PAM2 (PABPC1-interacting motif 2) motifs is assumed to bind to the PABC domain of PABPC1, on the poly(A) tail of mRNA. PAM2 motifs are also found in the major deadenylases Caf1–Ccr4 and Pan2–Pan3, whose activities are enhanced upon PABPC1 binding to these motifs. Their deadenylase activities are regulated by eRF3, in which two overlapping PAM2 motifs competitively prevent interaction with PABPC1. However, it is unclear how these overlapping motifs recognize PABC and regulate deadenylase activity in a translation termination-coupled manner. We used a dominant-negative approach to demonstrate that the N-terminal PAM2 motif is critical for eRF3 binding to PABPC1 and that both motifs are required for function. Isothermal titration calorimetry (ITC) and NMR analyses revealed that the interaction is in equilibrium between the two PAM2–PABC complexes, where only one of the two overlapping PAM2 motifs is PABC-bound and the other is PABC-unbound and partially accessible to the other PABC. Based on these results, we proposed a biological role for the overlapping PAM2 motifs in the regulation of deadenylase accessibility to PABPC1 at the 3′ end of poly(A).