PABPC1 SUMOylation enhances cell survival by promoting mitophagy through stabilizing U-rich mRNAs within stress granules

Abstract

Stress granules (SGs) are cytoplasmic, membraneless organelles that modulate mRNA metabolism and cellular adaptation under stress, yet the mechanisms by which they regulate cancer cell survival remain unclear. Here, we identify Poly(A)-Binding Protein Cytoplasmic 1 (PABPC1), a core SG component, as stress-inducible SUMOylation target. Upon various stress conditions, SUMOylated PABPC1 promotes SG assembly and enhances cancer cell survival. Transcriptome-wide analysis reveals that SUMOylated PABPC1 selectively stabilizes mRNAs enriched in conserved U-rich elements. Mechanistically, SUMOylated PABPC1 interacts with RNA-binding protein TIA1 to form PABPC1-SUMO-TIA1 complex that recruits U-rich mRNAs into SGs, protecting them from degradation. This process facilitates the expression of U-rich genes, such as mitophagy-related genes FUNDC1, BNIP3L, thereby maintaining cellular homeostasis and promoting cell survival under adverse conditions. Our findings reveal that PABPC1 SUMOylation connects stress granule assembly with selective U-rich mRNA stabilization and mitophagy, promoting cancer cell stress adaptation.

Fig. 1. PABPC1 is dynamically SUMOylated under stresses.

a Myc-PABPC1 and Flag-Ubc9 were transfected with His-SUMO1, His-SUMO2 or His-SUMO3 into HEK-293T cells and the Ni²⁺-NTA pulldown assay was performed to detect SUMOylation of PABPC1. b HA-PABPC1, His-SUMO1 and Flag-Ubc9 were transfected with or without EBG-SENP1 into HEK-293T cells as indicated and SUMOylation of PABPC1 was accessed by Ni²⁺-NTA pulldown assay. c Denaturing immunoprecipitation (IP) was conducted to evaluate endogenous SUMOylation of PABPC1 in HEK-293T^SENP1–/– Cells. d GST pulldown assay was performed to validate the SUMOylation of PABPC1 in E. coli based in vitro system. e The levels of SUMOylation of PABPC1 were assessed after a time course treatment with AS in HEK-293T cells. f Immunofluorescence staining was conducted to evaluate the formation of PABPC1 (green) and G3BP1(red) foci at different time points after AS treatment. Scale bar represented 10 μm, red arrows indicate foci formed by PABPC1 and G3BP1. g The SUMOylation levels of PABPC1 were examined in HEK-293T cells treated with AS for 1 h, followed by recovery for various durations. h Immunofluorescence staining was performed to monitor the changes in PABPC1 and G3BP1 foci following AS stimulation for 1 h and subsequent recover for different time points in H1299 cells. Scale bar represented 10 μm, red arrows indicate foci formed by PABPC1 and G3BP1. All results were shown with one representative image from three independent experiments (a-h).

Fig. 2. K512 is the major SUMOylation site of PABPC1.

a Identification of SUMOylation sites of PABPC1 in HEK-293T cells transfected with plasmids containing individual or combined mutations at K324, K333, and K512. Ni²⁺-NTA pulldown assays detected SUMOylation of PABPC1 in H1299 shPABPC1 (b) or H1299^{PABPC1–/–} (c) cells re-expressing Myc-PABPC1-WT/K512R. d Denaturing immunoprecipitation (IP) was conducted to evaluate endogenous SUMOylation of PABPC1 in H1299^{PABPC1–/–} cells re-expressing Myc-PABPC1-WT/K512R. e GST pulldown assay was performed to investigate SUMOylation of PABPC1-WT or PABPC1-K512R in prokaryotic expression systems. f Myc-PABPC1-WT/K512R, His-SUMO1, and Flag-Ubc9 were transfected into HEK-293T cells, and the SUMOylation levels of PABPC1 were detected after treatment with AS for 1 and 3 h. g Protein structural modeling of PABPC1 and distribution of its SUMOylation sites. * indicates the SUMO modification band at the K512 site of PABPC1. All western blot data were shown with one representative image from three independent experiments (a–f).

Fig. 3. PABPC1 SUMOylation promotes stress granule formation.

a Immunofluorescence staining of PABPC1 and G3BP1 in H1299^SENP1+/+ or H1299^SENP1–/– cells under AS-mediated stress condition. Scale bar represented 20 μm and statistical graphs showing the number of SGs. b Immunofluorescence staining of GFP-tagged PABPC1 and G3BP1 in H1299^{PABPC1–/–} cells re-expressing GFP-PABPC1-WT/K512R treated with AS for difference concentration. SGs foci were quantified by CellProfiler software and presented with dot graph. Scale bar represented 20 μm. c Immunofluorescence staining of GFP-tagged PABPC1 and G3BP1 in H1299^{PABPC1–/–} cells re-expressing GFP-PABPC1-WT/K512R treated with AS for 1 h and recovery for indicated time. Statistical graphs showing the number of SGs. Scale bar represented 20 μm. d Immunofluorescence staining of GFP-tagged PABPC1 and G3BP1 in H1299^PABPC1-/- cells re-expressing GFP-PABPC1-WT/K512R treated with AS for 1 h and 2-D08 (pan SUMOylation inhibitor) for difference concentration for 24 h. Statistical graphs showing the number of SGs. Scale bar represented 20 μm. e FRAP analysis of GFP-tagged PABPC1-WT/K512R in H1299^SENP-/- cells. The dashed circle with 0.8 μm diameter inside a large droplet was selected for photobleaching. Relative fluorescence intensity plotted as line graph over time. Mobile fraction was showed as bar graph, mean ± SD, n = 3 independent droplets. f Workflow for the isolation of PABPC1 interacting SG proteins. Volcano plots showing the differences in PABPC1-bound SG proteins in H1299^{PABPC1–/–} cells re-expressing Myc-PABPC1-WT/K512R after 1 h of AS treatment (g) and a 90 min recovery (h). i Gene Ontology (GO) pathway enrichment analysis was performed using proteins that are 1.5-fold up-regulated in binding with PABPC1-WT under AS treatment, P-values were calculated by DAVID bioinformatics tools using Fisher’s Exact Test. The data in (a–e) were shown as one representative image from three independent experiments. For Immunofluorescence staining, Statistical data are presented as mean ± SD, n ≥ 100 cells; P-values were calculated using two-tailed Student’s t test (a–d). For mass spectrometry analysis, two samples each group, P-values were calculated by two-tailed Student’s t test (g, h).

Fig. 4. SUMOylation of PABPC1 promotes mRNA stability under stress.

a Flowchart of the 4sU pulse-chase sequencing (4sU-Seq) process. Cumulative distribution plot (b) and frequency distribution plot (c) of mRNA half-life in H1299^{PABPC1–/–} cells re-expressing Myc-PABPC1-WT/K512R under normal condition. Cumulative distribution plot (d) and frequency distribution plot (e) of mRNA half-life in H1299^{PABPC1–/–} cells re-expressing Myc-PABPC1-WT/K512R under AS-mediated stress condition. f KEGG pathway analysis of genes in the 4sU-Seq data that showing a twofold increase in half-life in H1299^{PABPC1–/–} cells re-expressing Myc-PABPC1-WT under stress condition, P-values were calculated by DAVID bioinformatics tools using Fisher’s Exact Test. g List of enriched genes in the Mitophagy pathway. h, i 4sU-RT-qPCR was performed to detect the stability of FUNDC1, BECN1, and BNIP3L mRNA in H1299^{PABPC1–/–} cells re-expressing Myc-PABPC1-WT/K512R under normal and stress conditions. Decay graphs were generated by applying the one-phase decay model. For Cumulative fraction analysis, P-values were calculated using a two-sided Mann–Whitney U test (b, d). For 4sU-RT-qPCR, data were presented as mean ± SD, n = 3 biologically independent replicates, P-values were determined by one-side sum-of-squares F test (h–j).

Fig. 5. SUMOylation of PABPC1 at K512 enhances its affinity with mRNA under stress.

a Cumulative fraction analysis of RIP-Seq for mRNA transcripts bound to PABPC1 in H1299^{PABPC1–/–} cells re-expressing Myc-PABPC1-WT under normal and AS-mediated stress conditions. Boxes indicate the median, 25th and 75th percentiles, the whiskers denote the minima and maxima. b The cumulative distribution diagram shows the mRNA transcripts bound to PABPC1 in H1299^{PABPC1–/–} cells re-expressing Myc-PABPC1-WT/K512R treated with AS. Boxes indicate the median, 25th and 75th percentiles, the whiskers denote the minima and maxima. c Venn diagram analysis of mRNA transcripts bound to PABPC1 in the three groups of cells used in (a) and (b). WT_NT: transcripts bound to PABPC1 in H1299^{PABPC1–/–} cells re-expressing Myc-PABPC1-WT under normal condition; WT_AS: transcripts bound to PABPC1 in H1299^PABPC1-/- cells re-expressing Myc-PABPC1-WT under AS-mediated stress condition; K512R_AS: transcripts bound to PABPC1 in H1299^{PABPC1–/–} cells re-expressing Myc-PABPC1-K512R treated with AS. d Scatter plots analysis of mRNA transcripts with at least 1.5-fold alteration in binding to PABPC1 in the intersection of the data from (c). e oligo(dT) pulldown was performed in HEK-293T cells transfected with Myc-PABPC1-WT/K512R, His-SUMO1, Flag-Ubc9 and EBG-SENP1 to detect the effect of SUMOylation on the binding of PABPC1 to poly(A) RNAs. f RIP-qPCR detecting the binding of FUNDC1, BNIP3L, and BECN1 mRNAs to PABPC1 in H1299^PABPC1-/- cells re-expressing Myc-PABPC1-WT/K512R under normal and AS-mediated stress conditions. Cumulative distribution plot (g) and Heatmap (h) displaying mRNA half-life for transcripts recruited to PABPC1 with more than 1.5-fold up-regulated in WT_AS group compared to K512R_AS group; Boxes indicate median, 25th and 75th percentiles, and whiskers extend to 1.5 times the interquartile range (g). For Cumulative fraction analysis, P-values were calculated using a two-sided Mann–Whitney U test (a, b, g). For RIP-qPCR, presented as mean ± SD, n = 3 biologically independent replicates, P-values were determined by one-way ANOVA (f). Western blot data were shown with one representative image from three independent experiments (e).

Fig. 6. The PABPC1-SUMO-TIA1 complex recruits U-rich mRNAs to SGs.

a Motif discovery of transcripts in sequencing data using MEME software. Top: Motif analysis of transcripts exhibiting a 1.5-fold increase in binding to PABPC1-WT compared to PABPC1-K512R in RIP-Seq data under stress condition; Middle: Motif analysis of transcripts with a twofold up-regulated in mRNA half-life in H1299^{PABPC1–/–} cells re-expressing Myc-PABPC1-WT under stress condition, based on 4sU-Seq data; Bottom: Motif analysis of transcripts identified at the intersection of the above two datasets. b Cumulative fraction analysis of U-rich mRNA transcripts bound to PABPC1 in H1299^{PABPC1–/–} cells re-expressing Myc-PABPC1-WT under AS-mediated stress conditions, based on RIP-Seq data. c Co-Immunoprecipitation (Co-IP) detecting the interaction between PABPC1 and TIA1 in HEK-293T cells co-trasfected with His-SUMO1, Flag-Ubc9, and EBG-SENP1. d Co-Immunoprecipitation (Co-IP) accessing the interaction between PABPC1 and TIA1 in H1299^{PABPC1–/–} cells re-expressing Myc-PABPC1-WT/K512R under AS-mediated stress condition. e Co-Immunoprecipitation (Co-IP) showing the potential interaction between PABPC1 and several SIM mutated TIA1 in HEK-293T cells co-transfected with His-SUMO1 and Flag-Ubc9 plasmids. f RIP-qPCR detecting the binding of FUNDC1, BNIP3L, and BECN1 mRNAs to TIA1 under normal and stress conditions. g In vitro poly(U) RNA binding assay to detect the directly interaction of purified His-GFP-PABPC1 or His-GFP-PABPC1 + pE1E2S1, GST-TIA1, and Bio-poly(U) RNA. h Schematic diagram of TIA1 and PABPC1 binding to FUNDC1 mRNA. For Cumulative fraction analysis, P-values were calculated using a two-sided Mann–Whitney U test; in box plots, the lines represent the median, 25th and 75th percentiles, the whiskers denote the minima and maxima (b). For RIP-qPCR, data were presented as mean ± SD, n = 3 biologically independent replicates, P-values were determined by two-tailed unpaired t test (f). Western blot data were shown with one representative image from three independent experiments (c–e, g).

Fig. 7. SUMOylation of PABPC1 enhance mitophagy to promotes cell survival under stress condition.

a CCK-8 assay assessing proliferation of H1299^{PABPC1–/–} cells re-expressing Myc-PABPC1-WT/K512R under normal condition. CCK-8 (b) and plate colony formation (c) assay evaluating cell viability and clonogenic ability of H1299^{PABPC1–/–} cells re-expressing Myc-PABPC1-WT/K512R upon AS treatment. d Cell viability of H1299 and H1299^SENP1–/– cells overexpressing Myc-PABPC1-WT/K512R cells treated with 20 μM AS for 48 h. e Cell viability of H1299^{PABPC1–/–} cells re-expressing Myc-PABPC1-WT/K512R treated with 20 μM AS and different concentration of 2-D08 for 48 h. f qPCR quantification of FUNDC1, BNIP3L, and BECN1 mRNA levels in H1299^{PABPC1–/–} cells re-expressing Myc-PABPC1-WT/K512R under AS-induced stress. g Western blotting analysis of FUNDC1, BNIP3L, and BECN1 protein levels in H1299^{PABPC1–/–} cells re-expressing Myc-PABPC1-WT/K512R under AS treatment at indicated time points. h Mitophagy flux measured by mtKeima-based flow cytometry in H1299^{PABPC1–/–} cells re-expressing Myc-PABPC1-WT/K512R under AS treatment. i Live cell confocal imaging of mito-Keima detects mitophagy. Red mtKeima signal marks mitophagy within lysosomes. Scale bar, 20 μm. j qPCR quantification of mitochondrial DNA to nuclear DNA ratio in H1299^{PABPC1–/–} cells re-expressing Myc-PABPC1-WT/K512R under AS treatment. k Mitophagy flux determined by mito-Keima FACS in H1299^{PABPC1–/–} cells re-expressing Myc-PABPC1-WT/K512R with FUNDC1 knockdown under stress condition. CCK-8 assay (l) and plate colony formation (m) experiments detecting cell viability and clonogenic ability of H1299^{PABPC1–/–} cells re-expressing Myc-PABPC1-WT/K512R with FUNDC1 knockdown. n Determination of mitophagy flux using mito-keima FACS assay in H1299^{PABPC1–/–} cells re-expressing Myc-PABPC1-WT/K512R, as well as H1299^{PABPC1–/–} cells re-expressing Myc-PABPC1-K512R with FUNDC1 overexpression under stress condition. CCK-8 (o) and plate colony formation (p) assay evaluating cell viability and clonogenic ability of H1299^{PABPC1–/–} cells re-expressing Myc-PABPC1-WT/K512R and H1299^{PABPC1–/–} cells re-expressing Myc-PABPC1-K512R with FUNDC1 overexpression upon AS treatment. CCK-8 data and plate colony assay are presented as mean ± SD, n = 6 (a), 4 (b, d, e, l, o) or 5 (c, m, p) biological replicates, P-values were calculated by two-way ANOVA. qPCR (f, j) and FACS (P3 cells) (h, k, n) data plotted as mean ± SD of three biological replicates; P-values determined by one-way ANOVA. Western blot and Immunofluorescence staining data were representative of at least 3 biological repeats (g, i).

Fig. 8. Model illustrating the role of SUMOylated PABPC1 in stress tolerance.

Under stress conditions (e.g., sodium arsenite, heat shock, osmotic stress, glucose starvation), PABPC1 undergoes SUMOylation and interacts with TIA1 to form a PABPC1–SUMO–TIA1 complex. This complex recruits U-rich mRNAs into stress granules, preventing their degradation and promoting the expression of mitophagy-related genes such as FUNDC1, thereby maintaining cellular homeostasis and enhancing cancer cell stress adaptation.