Quantitative Characterization of Tob Interactions Provides the Thermodynamic Basis for Translation Termination-coupled Deadenylase Regulation

Lin Ruan,Masanori Osawa,Nao Hosoda,Shunsuke Imai,Asako Machiyama,Toshiaki Katada,Shin-Ichi Hoshino,Ichio Shimada

doi:10.1074/jbc.m110.138867

Abstract

Translation termination-coupled deadenylation is the first and often the rate-limiting step of eukaryotic mRNA decay in which two deadenylases, Ccr4-Caf1 and Pan2, play key roles. One of the deadenylases, Caf1, associates with Tob, which recruits Caf1 to the poly(A) tail through interactions with a cytoplasmic poly(A)-binding protein 1 (PABPC1). We previously proposed that the competition between Tob and eRF3 (a translation termination factor that interacts with PABPC1) is responsible for the regulation of deadenylase activity. However, the molecular mechanism of the regulation should be addressed by investigating the binding affinity and the cellular levels of these proteins. In this work, we characterized the human Tob interactions with Caf1 and a C-terminal domain of PABPC1 (PABC). Nuclear magnetic resonance (NMR) and Western blot analyses revealed that Tob consists of a structured N-terminal BTG-Tob domain and an unstructured C-terminal region with two conserved PAM2 (PABPC1-interacting motif 2) motifs. The BTG-TOB domain associates with Caf1, whereas the C-terminal PAM2 motif binds to PABC, with a K(d) value of 20 microM. Furthermore, we demonstrated that the levels of eRF3 and Tob in HeLa cells are 4-5 microM and less than 0.2 microM, respectively. On the basis of these results, we propose a thermodynamic mechanism for the translation termination-coupled deadenylation mediated by the Tob-Caf1 complex.

Highlights

The degradation of mRNA is an important step in the down-regulation of gene expression
We previously proposed that the competition between Tob and eRF3 is responsible for the regulation of deadenylase activity
This suggests that the interaction of PAM2-C and PABC is crucial for Tob-PABPC1 interaction [16]

Summary

EXPERIMENTAL PROCEDURES

Protein Expression and Purification—The cDNAs encoding full-length human Caf, Tob (full-length, residues 1–285, 1–148, 147–285, 121–148, and 260 –285), and the PABC domain of PABPC1 (residues 541– 623) were amplified by PCR using pCMVfrag-hCAF1, hTob, and PABPC1 constructs [16] as templates and cloned into the expression vector pGEX-6p-1 for Caf and Tob and pET42 for PABC. NMR Spectroscopy—For the backbone assignments of TobN148, uniformly 13C/15N-lableled TobN148 were prepared at a concentration of 0.3 mM in a buffer containing 10 mM phosphate (pH 6.0), 400 mM NaCl, 1 mM dithiothreitol, 0.5 M choline-O-sulfate, and 8% (v/v) D2O. To analyze the interactions between Tob fragments and PABC, aliquots of 408 –550 ␮M PABC solutions were injected into solutions of 29 –39 ␮M Tob fragments using the ITC buffer containing 10 mM NaH2PO4 and 50 mM NaCl (pH 7.0). Total cellular protein was isolated using buffer A (50 mM Tris-HCl (pH 6.8), 8% glycerol, 2% SDS, 2% 2-mercaptoethanol) and analyzed by Western blotting using anti-FLAG (Sigma), anti-PABPC1 (Abcam), anti-Tob (raised against His-tagged TobN285 protein), anti-Pan (raised against GST-Pan3(488 – 687) protein), or anti-eRF3 [18].

RESULTS

Tob Stoichiometry

NMR and Western blot analyses of