Fig 2: Functional analysis of total tRNA from NC and hypoxia groups’ testis in GC-2spd cells. A Schematic diagram of testis total tRNA-enriched fragment transfection into GC-2spd cells. B Nascent protein synthesis of GC-2spd cells transfected with total tRNA-enriched fragments from NC and Hypo group mouse testis. C Growth analysis of GC-2spd cells through counting cell number at 48 h after transfection and CCK8 at pointed time after transfection (n = 3~7, Additional file 13). D Expression levels of individual tRNAAla and tsRNAAla in GC-2spd cells transfected with total tRNA-enriched fragments from NC and Hypo group mouse testis after 48 h. E Western blot analysis of eIF4A and eIF4E expression in GC-2spd cells at 48 h transfected with total tRNA between NC and Hypo groups from mouse testis. **p < 0.01, ***p < 0.001, ****p < 0.0001; NB, northern blot. All results are shown as mean ± SEM. Statistical analysis was conducted using one-way ANOVA with uncorrected Fisher’s LSD

Fig 3: Influence of reconstituted eIF4F on translation termination. Relative peptide release rates (v0) at Nluc preTC induced by eRFs (8 nM each) in the presence of different combinations of eIF4F complex components eIF4G1, eIF4A, and eIF4E (35 nM each) (A) in the absence of ATP and (B) in the presence of ATP. p100, eIF4A, and eIF4E (20 nM each) (C) in the absence of ATP and (D) in the presence of ATP. (E) Relative peptide release rates (v0) at Nluc preTC induced by eRFs (8 nM each) in the absence and presence of eIF4B (15 nM) and eIF4G1 (35 nM)/eIF4F (5 nM). The data are shown as the mean ± standard error, number of repeats, n = 3. Asterisks indicate statistically significant differences between the values (*, P < .05; **, P < .01).

Fig 4: Effect of poly (A) tail, bound with PABP, on the activity of eIF4F components in translation termination. (A) The rates of peptide release (v0) at preTCs assembled on Nluc mRNA without poly (A) tail or with poly (A) tail. Peptide release was induced by eRF1 and eRF3a (8 nM each) in the presence of various concentrations of eIF4G1, eIF4A, eIF4E, and eIF4B. (B) Differences in peptide release rates at selected concentrations: eRFs 8 nM each, eIF4G1, eIF4A, eIF4E, and eIF4B 50 nM (1:6). RLU, relative luminescent units. The data are shown as the mean ± standard error, number of repeats, n = 3. Asterisks indicate statistically significant differences between the values (*, P < .05; **, P < .01; n.s., not significant).

Fig 5: Translation termination is stimulated by eIF4F. (A) Rate of peptide release (v0) at Nluc preTC induced by eRF1 and eRF3a (5 nM each) in the presence of various concentrations of eIF4F (left panel) and difference in peptide release rates at selected concentrations: eRFs 5 nM each and eIF4F 5 nM (1:1) (right panel). (B) Rate of peptide release (v0) at Nluc preTC induced by eRF1 (50 nM) in the presence of various concentrations of eIF4F (left panel) and difference in peptide release rates at selected concentrations: eRF1 50 nM and eIF4F 100 nM (1:2) (right panel). RLU, relative luminescent units. (C) Toe-print analysis of TC formation in the reconstituted mammalian translation system in the presence of eRF1 (AGQ) and eIF4F or eIF4G1. ru, relative units. (D) GTPase activity of eRF3a in the presence of various concentrations of eIF4F (left panel) and difference in the amount of the released 32Pi at selected concentrations: eRFs 166 nM each and eIF4F 312 nM (1:1.8) (right panel). The data are shown as the mean ± standard error, number of repeats, n = 3. Asterisks indicate statistically significant differences between the values (*, P < .05; **, P < .01).