The Drosophila Poly(A) Binding Protein-Interacting Protein, dPaip2, Is a Novel Effector of Cell Growth

Translation plays an important role in the regulation of gene expression and is implicated in the control of cell growth, proliferation, and differentiation. In eukaryotes, initiation is the rate-limiting step of translation in most circumstances and is a major target for regulation (reviewed in reference 18). The 5′ cap structure (m7GpppN, where m is a methyl group and N is any nucleotide) of the mRNA is recognized by the eukaryotic initiation factor 4F (eIF4F) complex. eIF4F is comprised of three subunits: (i) eIF4E, the cap binding protein; (ii) eIF4A, a bidirectional ATP-dependent RNA helicase; and (iii) eIF4G, a modular scaffolding protein, which possesses binding sites for eIF4E and eIF4A and recruits the 40S ribosomal subunit to the mRNA via its interaction with eIF3 (20). The 3′ poly(A) tail of the mRNA is bound by the poly(A) binding protein (PABP). PABP is a phylogenetically conserved protein that functions in mRNA stability and translation (48). PABP is an essential protein: in Saccharomyces cerevisiae, deletion of the PAB1 gene is lethal (49) and a P-element insertion in the Drosophila melanogaster PABP gene is embryonic lethal (53). PABP is an ∼630-amino-acid (aa) protein containing four RNA recognition motifs (RRMs) arranged in tandem and a proline-rich C-terminal domain (reviewed in reference 24). RRMs 1 and 2 are the major contributors to the poly(A) binding activity of PABP (10, 30). PABP directly interacts with eIF4G, leading to circularization of the mRNA by bridging the 5′ and 3′ extremities (closed-loop model) (40, 48). The closed-loop model explains the synergistic enhancement of translation by the 5′ cap structure and the 3′ poly(A) tail of the mRNA (15). By joining the 5′ and 3′ ends of the mRNA, circularization may facilitate recycling of ribosomes, initiation complex formation, or the 60S ribosome-joining step (24, 52). Our group has identified two human proteins that interact directly with PABP: Paip1 and Paip2 (PABP-interacting proteins 1 and -2). Paip1 stimulates, while Paip2 represses, translation (8, 27). Paip2 inhibits translation by reducing the binding of PABP to the poly(A) tail and by competing with Paip1 for binding to PABP. Paip1 and Paip2 share two conserved PABP-interacting motifs (PAMs). PAM1 consists of a stretch of acidic amino acids in the middle of Paip2 (aa 22 to 75) and at the C terminus of Paip1 (aa 440 to 479), and it binds strongly to RRMs 2 and 3 and to RRMs 1 and 2 of PABP, respectively (25, 45). The second binding site, PAM2, also called the PABP C-terminal binding motif, resides in the C terminus of Paip2 (aa 106 to 120) (25) and the N terminus of Paip1 (aa 123 to 137) (45). PAM2 consists of a short stretch of 15 aa and binds to the C terminus of PABP (within aa 546 to 619) with a lower affinity (∼10- and ∼200-fold for Paip1 and Paip2, respectively) than that of the PAM1-PABP interaction (25, 45). PAM2 is also found in several additional proteins, including eukaryotic release factor 3 (eRF3), ataxin 2, and transducer of ErbB-2 (Tob) (11, 29). Thus, Paip2 and Paip1 might compete with some of these PAM2 binding partners to regulate PABP function. To study the biological role of Paip2, the Drosophila homologue of the human Paip2 (hPaip2), dPaip2, was isolated and characterized. Its ability to interact with Drosophila PABP (dPABP), inhibit translation, and interfere with dPABP poly(A) binding activity was demonstrated. Importantly, dPaip2 inhibits growth in flies.