Quantitative characterization of Tob interactions provides the thermodynamic basis for translation termination-coupled deadenylase regulation.
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.
Ruan
Lin
L
Department of Physical Chemistry, Graduate School of Pharmaceutical Sciences, The University of Tokyo, Tokyo 113-0033, Japan.
Osawa
Masanori
M
Hosoda
Nao
N
Imai
Shunsuke
S
Machiyama
Asako
A
Katada
Toshiaki
T
Hoshino
Shin-ichi
S
Shimada
Ichio
I
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D001665:Binding Sites; D002151:Calorimetry; D055598:Chemical Phenomena; D006367:HeLa Cells; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins / Q000737:chemistry / Q000378:metabolism*; D042541:Intracellular Space / Q000378:metabolism; D009682:Magnetic Resonance Spectroscopy; D010446:Peptide Fragments / Q000378:metabolism; D010454:Peptide Termination Factors / Q000378:metabolism; D039122:Poly(A)-Binding Protein I / Q000737:chemistry / Q000378:metabolism*; D011485:Protein Binding; D014176:Protein Biosynthesis; D017510:Protein Folding; D017434:Protein Structure, Tertiary; D012333:RNA, Messenger / Q000378:metabolism; D013816:Thermodynamics; D014157:Transcription Factors / Q000378:metabolism*; D025521:Tumor Suppressor Proteins / Q000737:chemistry / Q000378:metabolism*
C489780:CNOT8 protein, human; D047908:Intracellular Signaling Peptides and Proteins; D010446:Peptide Fragments; D010454:Peptide Termination Factors; D039122:Poly(A)-Binding Protein I; D012333:RNA, Messenger; C098669:TOB1 protein, human; D014157:Transcription Factors; D025521:Tumor Suppressor Proteins; C087971:peptide-chain-release factor 3