Paip1 Interacts with Poly(A) Binding Protein through Two Independent Binding Motifs

Abstract

The 3′ poly(A) tail of eukaryotic mRNAs plays an important role in the regulation of translation. The poly(A) binding protein (PABP) interacts with eukaryotic initiation factor 4G (eIF4G), a component of the eIF4F complex, which binds to the 5′ cap structure. The PABP-eIF4G interaction brings about the circularization of the mRNA by joining its 5′ and 3′ termini, thereby stimulating mRNA translation. The activity of PABP is regulated by two interacting proteins, Paip1 and Paip2. To study the mechanism of the Paip1-PABP interaction, far-Western, glutathione S-transferase pull-down, and surface plasmon resonance experiments were performed. Paip1 contains two binding sites for PABP, PAM1 and PAM2 (for PABP-interacting motifs 1 and 2). PAM2 consists of a 15-amino-acid stretch residing in the N terminus, and PAM1 encompasses a larger C-terminal acidic-amino-acid-rich region. PABP also contains two Paip1 binding sites, one located in RNA recognition motifs 1 and 2 and the other located in the C-terminal domain. Paip1 binds to PABP with a 1:1 stoichiometry and an apparent K_d of 1.9 nM.

We thank C. Lister and C. Binda for excellent technical assistance and Y. Svitkin, H. Imataka, J. Berlanga, A. Brasey, and J. Dostie for helpful discussions. We thank A. Baass and D. Fantus for help in the preparation and purification of Paip1 deletion mutants. We thank M. Miron and J. Dostie for anti-GST antibody and H. Imataka for plasmid pcDNA3-GST.

This research was supported by a grant from the Canadian Institute of Health Research (CIHR). N.S. is a CIHR distinguished scientist and a Howard Hughes Medical Institute International Scholar. G.R., K.K., and A.K. are recipients of predoctoral studentships from the CIHR. G.R. is the recipient of a McGill Major studentship. G.D.C. is supported by the Protein Engineering Network of Centres of Excellence.