Abstract
Programmed cell death 4 protein (PDCD4) regulates many vital cell processes, although is classified as a tumor suppressor because it inhibits neoplastic transformation and tumor growth. For example, PCDC4 has been implicated in the regulation of transcription and mRNA translation. PDCD4 is known to inhibit translation initiation by binding to eukaryotic initiation factor 4A and elongation of oncogenic c- and A-myb mRNAs. Additionally, PDCD4 has been shown to interact with poly(A)-binding protein (PABP), which affects translation termination, although the significance of this interaction is not fully understood. Considering the interaction between PABP and PDCD4, we hypothesized that PDCD4 may also be involved in translation termination. Using in vitro translation systems, we revealed that PDCD4 directly activates translation termination. PDCD4 stimulates peptidyl-tRNA hydrolysis induced by a complex of eukaryotic release factors, eRF1-eRF3. Moreover, in combination with the PABP, which also stimulates peptide release, PDCD4 activity in translation termination increases. PDCD4 regulates translation termination by facilitating the binding of release factors to the ribosome, increasing the GTPase activity of eRF3, and dissociating eRF3 from the posttermination complex. Using a toe-printing assay, we determined the first stage at which PDCD4 functions—binding of release factors to the A-site of the ribosome. However, preventing binding of eRF3 with PABP, PDCD4 suppresses subsequent rounds of translation termination. Based on these data, we assumed that human PDCD4 controls protein synthesis during translation termination. The described mechanism of the activity of PDCD4 in translation termination provides a new insight into its functioning during suppression of protein biosynthesis.
Highlights
Liver, and breast tumors, as well as glioblastomas [7,8,9,10]
PreTCs containing Nano luciferase (Fig. S1A) was fixed in the P-site by the mutant eukaryotic release factor 1 (eRF1)(AGQ), which is unable to hydrolyze peptidyltRNA to accomplish the process. These complexes were purified from the rabbit reticulocyte lysate (RRL) by sucrose density centrifugation at the high ionic strength, which resulted in eRF1(AGQ) dissociation from the ribosome
To address the possible effect of programmed cell death protein (PDCD4) in the complex with poly (A)-binding protein (PABP) on the translation termination process, eRF1, eRF3a, PDCD4, and PABP were added to the purified pretermination complex (preTC)(NL-polyA) simultaneously or in different combinations, and the luminescence was measured as a function of time
Summary
Discovery of a novel role of tumor suppressor PDCD4 in stimulation of translation termination. In combination with the PABP, which stimulates peptide release, PDCD4 activity in translation termination increases. PDCD4 regulates translation termination by facilitating the binding of release factors to the ribosome, increasing the GTPase activity of eRF3, and dissociating eRF3 from the posttermination complex. Preventing binding of eRF3 with PABP, PDCD4 suppresses subsequent rounds of translation termination. The N-terminus of PDCD4 interacts with poly (A)-binding protein (PABP), which is involved in maintaining mRNA stability, and implementing effective translation initiation and termination [25, 26]. PABP increases the binding efficiency of the human release factors to the ribosome through interactions with the N-terminal domain of eRF3a [26]. We show that PDCD4 stimulates translation termination and propose a model for its function
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