Fig 2: PKA signaling supports translation initiation.(A) Gene set enrichment analysis (GSEA) of significantly altered phosphoproteins following doxycycline (dox) induction of PKA in proteomics data from this study or chemical PKA stimulation in our prior publications. Results are shown for Hallmark gene sets, with statistically significant enrichment of altered phosphoproteins annotated to be involved in translation initiation. (B) Time course of forskolin (FSK)/3-isobutyl-1-methylxanthine (IBMX) in Colo741 and FLX1 showing increased phosphorylation of eIF4B Ser422. (C) Impact of 24 hr dox treatment on phosphorylation of eIF4B at Ser422 in engineered FLX1 cells with dox-inducible 3xFLAG-PRKAR1AG325D. (D) Immunoblots showing the change of PKA activity and PKAc and c-MYC expression and phosphorylation of eIF4B Ser422 in Colo741 and FLX1 cells after pooled PRKACA siRNA knockdown for 24, 48, and 72 hr vs. 72 hr with non-targeting control (NTC). Long and short exposures are used in FLX1 to show knockdown effect in wild type (WT) PKAc and the DNAJ-PKAc fusion. Figure 6—source data 1.Tables for Figure 6A. Figure 6—source data 2.Images for Figure 6B. Figure 6—source data 3.Images for Figure 6C. Figure 6—source data 4.Images for Figure 6D part 1/4. Figure 6—source data 5.Images for Figure 6D part 2/4. Figure 6—source data 6.Images for Figure 6D part 3/4. Figure 6—source data 7.Images for Figure 6D part 4/4.

Fig 3: Tethering of eIF4A1/2 to chromosomes accelerates chromatin decondensation.A Time-lapse images of H2B-mPlum-FKBP HeLa cells expressing EGFP-FRB-eIF4A1 after adding 200 nM DMSO (control, - rapamycin) or rapamycin. Time is normalized to the first anaphase frame. Scale bars: 10 µm. B Time-dependent quantitation of the chromatin area of the experiments as in (A), normalized to the first anaphase frame. Dots represent mean ± s.e.m. from 12 (-rapamycin) and 14 (+ rapamycin) cells. C Same as in (A) but with the ATPase deficient mutant of eIF4A1 E183Q. D Same as in (B) but with the ATPase deficient mutant of eIF4A1 E183Q from 7 (-rapamycin) and 9 (+ rapamycin) cells. E Same as in (A) but with the RNA-binding mutant of eIF4A1 R362Q. F Same as in (B) but with the RNA-binding mutant of eIF4A1 R362Q from 9 (-rapamycin) and 10 (+ rapamycin) cells. G Same as in (A) but with EGFP-FRB-eIF4A2. H Same as in (B) but with EGFP-FRB-eIF4A2 with 15 (-rapamycin) and 17 (+ rapamycin) cells. I Same as in (A) but with EGFP-FRB-eIF4A3. J Same as in (B) but with EGFP-FRB-eIF4A3 with 10 (+ rapamycin) and 10 (-rapamycin) cells. K Time-dependent quantification of the chromatin area normalized to the first anaphase frame. HeLa cells stably expressing H2B-mCherry were co-transfected with GFP or eIF4A3-GFP and 40 nM control or a combination of eIF4A1 and eIF4A2 siRNA oligos for 48 h. Dots represent mean ± s.e.m. from each condition with 53 (EGFP + siCtrl), 37 (EGFP + sieIF4A1/2), 71 (eIF4A3-EGFP + siCtrl) or 45 (eIF4A3-EGFP + sieIF4A1/2) daughter chromatin masses. L Western blot showing the downregulation of eIF4A1/2 at 48 h post-transfection with 40 nM siRNA oligos and EGFP or eIF4A3-EGFP in HeLa cells stably expressing H2B-mCherry. Samples were analyzed with antibodies recognizing human eIF4A1/2. Actin serves as loading control. Source data are provided as a Source Data file.

Fig 4: eIF4A1/2 is rapidly excluded from the reforming nucleus.A eIF4A1/2 immunostaining (green in overlay) of HeLa cells stably expressing H2B-mCherry (magenta in overlay) at mitotic different stages, representative for 4 independent experiments. Scale bars: 10 µm. B eIF4A1/2 immunostaining (green in overlay) of interphase HeLa cells stably expressing H2B-mCherry (magenta in overlay), untransfected or transfected with 20 nM control or a combination of eIF4A1 and eIF4A2 siRNA oligos for 72 h, representative for 4 independent experiments. Scale bars: 10 µm. C Fixed samples of different mitotic stages of HeLa cells expressing H2B-mCherry (magenta in overlay) and GFP or eIF4A1-EGFP (green in overlay), representative for 2 independent experiments. Scale bars: 10 µm. D Fixed samples of interphase HeLa cells expressing H2B-mCherry (magenta in overlay) and GFP or eIF4A1-EGFP (green in overlay), representative for 2 independent experiments. Scale bars: 10 µm. E Time-lapse images of H2B-mCherry HeLa cells expressing eIF4A1-EGFP, the corresponding ATPase deficient mutant eIF4A1 E183Q or the RNA-binding deficient R362Q mutant, EGFP, eIF4A2-EGFP, eIF4A3-EGFP, EGFP-eIF4B, EGFP-eIF4G1 or EGFP-eIF4H, representative for 3 independent experiments per construct. Time is normalized to the first anaphase frame. Scale bars: 10 µm.

Fig 5: Downregulation of eIF4A1/2 decelerates mitotic chromatin decondensation in cells.A Telophase duration in H2B-mCherry HeLa cells, transfected with 20 nM siRNA control or against PP2A, eIF4A1 (three different oligos), eIF4A2 or a combination of eIF4A1 (oligo A) and eIF4A2, analyzed by live cell imaging 48–63 h post-transfection. Mean telophase time of at least 150 mitotic events per condition from three independent experiments with the individual (triangles) and overall means ± s.e.m. Two-tailed unpaired t test with Welch’s correction (sieIF4A1/2, *P = 0.02). B Chromatin area analysis from (A) with at least 90 mitotic events per condition, normalized to the first anaphase frame. Lines represent means ± s.e.m. C Nuclear area of interphase cells, defined by 10 frames before interphase-prophase transition, from (A), 90 cells per condition. Means of three independent experiments (triangles) and overall mean ± s.e.m. with values for each transfection. Two-tailed unpaired t test with Welch’s correction (sieIF4A1-A, *P = 0.01). D eIF4A1/2 Western blot of H2B-mCherry HeLa cells, 72 h post-transfection with 20 nM siRNA oligos, corresponding to (A–C), with eIF4A1 antibodies (upper) or antibodies recognizing eIF4A1 and eIF4A2 (lower panel). Actin serves as loading control. Signal intensity, normalized to actin, of four independent experiments (triangles) and overall mean ± sd. Two-tailed unpaired t test with Welch’s correction (eIF4A1, ***P = 6.73 × 10−4; eIF4A1/2, **P = 3.58 × 10−3). E Time-lapse images of H2B-mCherry HeLa cells, transfected with 20 nM control or a combination of eIF4A1 and eIF4A2 siRNA oligos for 72 h, normalized to the first anaphase frame. F Analysis of the experiments as in (E). Dots represent mean ± s.e.m. from 11 (siCtrl) and 10 (sieIF4A1/2) cells per condition. G Chromatin area quantification of H2B-mCherry HeLa cells, co-transfected with eIF4A1-EGFP or siRNA resistant eIF4A1-EGFP and 40 nM control or a combination of eIF4A1 and eIF4A2 siRNA oligos for 48 h, normalized to the first anaphase frame. Dots represent mean ± s.e.m. from each condition with 47 (eIF4A1-EGFP+ siCtrl), 57 (eIF4A1-EGFP + sieIF4A1/2), 72 (eIF4A1res-EGFP + siCtrl) or 43 (eIF4A1res-EGFP + sieIF4A1/2) daughter chromatin masses. H eIF4A1/2 western blot of H2B-mCherry HeLa cells, 48 h post-transfection with 40 nM siRNA oligos and eIF4A1-EGFP or eIF4A1res-EGFP. Actin serves as loading control. Source data are provided as a Source Data file. Scale bars: 10 µm.