RNA Structures Called rG4 Accelerate Cellular Aging by Stalling Protein-Making Machinery

Cellular senescence—a state of irreversible growth arrest—drives tissue aging and age-related disease partly through the collapse of protein homeostasis (proteostasis). While ribosome pausing is known to contribute to proteostasis failure, the molecular triggers linking it to senescence were poorly understood. This study identifies RNA G-quadruplex (rG4) structures as a previously unrecognized driver of ribosome stalling in senescent cells.

Using parallel RNA-seq and Ribo-seq (ribosome profiling) on young proliferative versus replicatively senescent human BJ fibroblasts, the authors found that genes with reduced translational efficiency (TE) outnumbered those with increased TE by roughly four-to-one in senescent cells. Critically, the largest affected gene cluster showed changes exclusively at the translational level—not at mRNA abundance—implicating translation elongation rather than transcription. Ribosome pause score analysis revealed that pausing was enriched immediately upstream of guanine-rich motifs, and rG4 structure prediction algorithms confirmed these sequences had significantly higher G-quadruplex-forming potential than shuffled controls.

To establish causality, the team performed G4 RNA immunoprecipitation sequencing (rG4-RIP-seq) and immunofluorescence with G4-binding probes (G4P and BG4). In young cells, rG4 signal was predominantly nuclear, but in senescent cells it expanded dramatically into the cytoplasm—co-localizing with ribosomes—indicating cytoplasmic rG4 formation in mRNA is a senescence-associated event. Reporter assays confirmed that inserting rG4-forming sequences into coding regions (CDS) was sufficient to impede translation both in cell-based and cell-free systems, while mutating these sequences rescued translation efficiency.

The study then identified DHX9, an RNA helicase with known rG4-unwinding activity, as a key regulator. DHX9 expression was significantly reduced in senescent BJ cells, aged mouse tail-tip fibroblasts, and tissues from aged mice. Overexpressing DHX9 in senescent cells reduced rG4 abundance and alleviated ribosome pausing, while DHX9 knockdown in young cells phenocopied the senescent state—increasing rG4 levels, promoting ribosome stalling, disrupting proteostasis, and accelerating senescence markers including SA-β-galactosidase activity, p21 upregulation, and SASP factor secretion. Conversely, pharmacological stabilization of rG4 structures (using the G4 ligand PDS) worsened senescence, reinforcing the functional significance of rG4 accumulation.

These findings establish a mechanistic axis: declining DHX9 → rG4 accumulation in mRNA CDS → ribosome pausing → reduced TE for key proteins → proteostasis collapse → exacerbated senescence. The conserved observation in aged mouse tissues suggests this pathway operates in vivo during mammalian aging, making DHX9 and rG4 resolution potential targets for interventions aimed at slowing cellular and organismal aging.