Fig 2: Minimal required components of translation termination apparatus to be activated by PDCD4.A, TC formation analysis with eRF1 (AGQ)+eRF3a/eRF3c in the presence/absence of PDCD4 on MVHL mRNA and MVHL-poly(A). The background was subtracted and each mean value was normalized to the value of eRF1 (AGQ)+eRF3a/eRF3c. The black dots show the replicate values. The error bars represent the standard error, ∗∗p < 0.01, t test, ∗p < 0.05, t test. B, Termi-Luc peptide release assay showing level of peptidyl-tRNA hydrolysis induced by eRF1+eRF3c in the presence/absence of PDCD4. Time progress curves showing luminescence (in relative luminescence units, RLU) with NLuc luciferase released from ribosome complex upon treatment with the proteins of interest (n = 3). The error bars represent the standard mean deviation. C, TC formation analysis with eRF1 (AGQ) in the presence/absence of PDCD4 or GST as a negative control on MVHL mRNA, MVHL-poly(A) or MVHL-poly(A)+PABP. D, Termi-Luc peptide release assay showing level of peptidyl-tRNA hydrolysis induced by eRF1 in the presence/absence of PDCD4 (n = 3).

Fig 3: PDCD4 enhances translation termination independently of PABP.A, interaction scheme of PDCD4, PABP, eIF4AI domains. B, Termi-Luc peptide release assay in the presence/absence of PDCD4, PABP, and the release factors. Time progress curves showing luminescence (in relative luminescence units, RLU) with NLuc luciferase released from ribosome complex upon treatment with the proteins of interest (n = 3). The error bars represent the standard mean deviation.

Fig 4: Effect of eIF4A on PDCD4 activity in translation termination.A, TC formation analysis eRF1(AGQ)+eRF3a in the presence of eIF4A and PDCD4. The black dots show the replicate values. B, GTPase activity of eRF3c in presence eIF4A and PDCD4 (n = 3). The background was subtracted and each mean value was normalized to the value of eRF1(AGQ)+eRF3a or eRF1+eRF3c activity. The error bars represent the standard errors, stars (∗∗) mark a significant difference from the respective control p < 0.01, t test. C, effect of eIF4A on PDCD4-mediated peptide release in the presence of eRF1 and eRF3a (n = 3); time progress curves showing luminescence (in relative luminescence units, RLU) with NLuc luciferase released from ribosome complex upon treatment with the proteins of interest. The error bars represent the standard mean deviation.

Fig 5: Activity of domains and mutant forms of PDCD4 in translation termination.A, scheme of PDCD4 point mutants and domains. B, TC formation efficiency in presence of eRF1 (AGQ)+eRF3a and PDCD4 or MA3m-c domain of PDCD4. C, Termi-Luc peptide release assay in the presence of eRF1+eRF3a and MA3m, Ma3c, MA3m-c domains of PDCD4 (n = 3). D, GTPase assay in the presence of 80S subunits and eRF1+eRF3c with PDCD4 and MA3m, MA3c or MA3m-c domains. E, Termi-Luc peptide release assay in the presence of eRF1+eRF3a and PDCD4 wt or PDCD4 point mutants: E52A, K58E, K58A, D414A, and D418A (n = 2). For TC formation and GTPase analysis, the background was subtracted and each mean value was normalized to the value of eRF1 (AGQ)+eRF3a/eRF3c. The black dots show the replicate values. The error bars represent the standard error, ∗∗p < 0.01, t test, for peptide release analysis: time progress curves showing luminescence (in relative luminescence units, RLU) with NLuc luciferase released from ribosome complex upon treatment with the proteins of interest (n = 2). The error bars represent the standard mean deviation.