RNA G-quadruplex (rG4) exacerbates cellular senescence by mediating ribosome pausing

Figure 1.. Ribosome pausing-reduced TE is enriched in G-rich regions in senescent cells. (A) Schema illustrating the workflow for analyzing TE in young (proliferative stage) and replication senescent cells. (B) Volcano plot illustrating changes in translational efficiency between young and senescent BJ cells, with cutoffs set at P < 0.05 and |log2TE-fold change| ≥ 1. (C) Intersection analysis of genes with different patterns of change in transcriptome and translatome and genes with downregulated TE. The intersection analysis among these uses the Inclusive intersection mode in ComplexUpSet. (D) Cumulative frequency histogram of ribosome pause scores in the CDS of senescent and young BJ cells. (E) Relative ribosome pausing during cellular senescence. The colored points indicate codon positions with significantly increased pausing (fold change ≥ 2, P value < 0.05), all other translatome positions in gray. Statistical analysis was performed using a two-sided Wilcoxon test. (F) Enriched motifs identified in sequences located a 20-nt region downstream of RPScore upregulated site using the Homer software. The frequency of occurrence for each motif across all input sequences has also been included…

Figure 2.. RNA G-quadruplex signal is increased in senescent cells and hinders translation. (A) Cytoplasm G4P signal was increased in young and replication senescent G4P-cells. G4P was induced by DOX and IF was performed with Flag antibody. Cells contain more than one cytoplasm G4P foci were calculated (n ≥ 100 cells). All values are mean ± SEM of more than three independent experiments. (B) Cytoplasm BG4 signal was increased in replication senescent cells. IF was performed with BG4 and Flag antibody. Cells contain more than one cytoplasm BG4 foci were calculated (n ≥ 100 cells). All values are mean ± SEM of more than three independent experiments. (C) rG4 was increased in replication senescent cells. Imaging of replication senescent cells stained with QUMA-1 (2 μmol/L) for 20 min at 37°C. Cells contain more than one cytoplasm QUMA-1 foci were calculated (n ≥ 100 cells). All values are mean ± SEM of more than three independent experiments. (D) Most intersection genes between Ribo-seq and G4P-RIP-seq have decreased TE in senescent cell. (E) G4P-RIP-seq enrich high-fold change genes in senescent cell. (F) According to (D), the protein TE of rG4-enriched genes is decreased. (G) IGV depicting…

Figure 3.. RNA G-quadruplex exacerbates ribosome pausing leading to translation impairment. (A) Schematic diagram of the dual fluorescence reporter. (B) Schematic diagram of the primer design. (C) Random and rG4 sequences not affect the dual fluorescence reporter transcription. mRNA levels were quantified by qPCR. (D) Fluorescence microscopy and mCherry+/GFP− puncta formation in BJ fibroblast cells harboring the dual fluorescence reporter. BJ cells were transfected with dual fluorescence reporter for 72 h and treated with cPDS (2 μmol/L). (E) Quantification of (D). Cells contain mCherry+/GFP− puncta were calculated (n ≥ 50 cells). All values are mean ± SEM of more than three independent experiments. (F) CFS. According to (A), use CFS to generate mCherry-random-GFP fusion protein or mCherry-rG4-GFP fusion protein in vitro. (G) Immunoblot of dual fluorescence reporter in 293T cells. 293T cells were transfected with dual fluorescence reporter for 72 h and treated with cPDS (2 μmol/L). Western was performed with indicated antibodies. Truncated and full-length products are noted. n = 3 biological replicates with representative example shown. (H) Immunoblot analysis of indicated proteins in BJ…

Figure 4.. Stabilization of RNA G-quadruplex accelerates cellular senescence. (A) Fluorescence microscopy and mCherry+/GFP− puncta formation in young and senescent BJ fibroblast cells harboring the dual fluorescence reporter. Young and senescent BJ cells were transfected with dual fluorescence reporter for 72 h. (B) Quantification of (A). Cells contain mCherry+/GFP− puncta were calculated (n ≥ 50 cells). All values are mean ± SEM of more than three independent experiments. (C) cPDS increased rG4 in presenescent cells. PD36 BJ cells were treated with cPDS (2 μmol/L) for 48 h. IF was performed with BG4 and Flag antibodies. (D) Quantification of (C). Cells contain more than one cytoplasm BG4 foci were calculated (n ≥ 50 cells). All values are mean ± SEM of more than three independent experiments. (E) Stabilization of rG4 increased SA-β-Gal positive cells. (F) Quantification of (E). SA-β-Gal positive cells were calculated (n ≥ 100 cells). All values are mean ± SEM of more than three independent experiments. (G) KEGG pathway enrichment analysis of differentially enrich genes between DMSO and cPDS treatment BJ cells. (H) Immunoblot analysis of indicated proteins in presenescent BJ cells after…

Figure 5.. DHX9 deficiency facilitates RNA G-quadruplex stability and promotes cellular senescence. (A) Veen diagram representing the overlapping genes of DEAD box and DEATH box RNA helicase identified through RNA-seq and Ribo-seq. Only DHX9 and DDX19B showed decreased mRNA and translation levels in senescent cells. (B) DHX9 deficiency accelerates cellular senescence. BJ cells were treated with VP-16 (40 μmol/L, 24 h) and transfected with siRNAs (72 h), then stained by SA-β-Gal. (C) Quantification of (B). SA-β-Gal positive cells were calculated (n ≥ 100 cells). All values are mean ± SEM of more than three independent experiments. (D) Immunoblot analysis of indicated proteins in young and senescent BJ cells. (E) rG4 was increased in DHX9 deficiency cells. BJ cells were transfected with siRNAs for 72 h. Imaging of DHX9 deficiency cells stained with QUMA-1 (2 μmol/L) for 20 min at 37°C. (F) Quantification of (E). Cells contain more than one cytoplasm QUMA-1 foci were calculated (n ≥ 100 cells). All values are mean ± SEM of more than three independent experiments. (G) Fluorescence microscopy and mCherry+/GFP− puncta formation in DHX9 deficiency BJ fibroblast cells harboring the dual…

Figure 6.. Disruption of proteostasis accompanied by reduced DHX9 level and increased rG4 abundance in aged mice. (A) Schematic diagram of organs isolation. (B) Immunohistochemical analysis of DHX9 expression in Liver and Lung. (C and D) Quantification of (B). DHX9 positive cells were calculated. All values are mean ± SEM of more than three mice. (E) Immunohistochemical analysis of DHX9 expression in Heart, Spleen, and Kidney. (F–H) Quantification of (E). DHX9 positive cells were calculated. All values are mean ± SEM of more than three mice. (I) Schematic diagram of Tail-tip fibroblast cells isolation. (J) Immunoblot analysis of indicated proteins in young and aged Tail-tip fibroblast cells. (K) Quantification of (J). Relative protein level of DHX9. (L) rG4 was increased in aged mice Tail-tip fibroblast cells. Imaging of Tail-tip fibroblast cells stained with QUMA-1 (2 μmol/L) for 20 min at 37°C. (M) Quantification of (L). Cells contain more than one cytoplasm QUMA-1 foci were calculated (n ≥ 100 cells). All values are mean ± SEM of more than three independent experiments. (N) Puromycin incorporation assay using TFB cells shows reduced Puromycin incorporation in 24 months TFB. The right…

Figure 7.. Proposed working model for RNA G-quadruplex to regulate cellular senescence. DHX9 functions to unwind mRNA rG4 structures, thereby preventing ribosome pausing during translation. In senescent cells, a decrease in the abundance of DHX9 protein results in the persistent presence of mRNA rG4 structures, leading to the halting of ribosomes. Consequently, the stalled ribosomes lead to collisions, ultimately contributing to the collapse of proteostasis. This figure was created with photoshop.