Introduction
Cellular senescence plays a significant role in the aging of organs and organisms, as the accumulation of senescent cells is closely associated with the aging process and the degeneration of various tissues and organs (
Gorgoulis et al., 2019). Furthermore, the imbalance in proteostasis, a fundamental characteristic of cellular senescence, is evident in numerous age-related diseases (
Lopez-Otin et al., 2013;
Montague-Cardoso, 2021;
Rubinsztein et al., 2011;
Salminen et al., 2012). Neurodegenerative disorders, for instance, are characterized by the abnormal aggregation of proteins (
Galluzzi et al., 2018;
Godin et al., 2016;
Wilson et al., 2023). Protein synthesis and degradation are subject to precise regulation within cellular contexts. When errors occur during these processes, cellular mechanisms responsible for maintaining protein quality control are activated. These mechanisms include the unfolded protein response (UPR;
Senft and Ronai, 2015), the ubiquitin-proteasome system (
Pohl and Dikic, 2019), and the autophagy-lysosome pathway (
Sha et al., 2017). The principal aim of these response mechanisms is to prevent the excessive accumulation of misfolded or unassembled peptides by facilitating the degradation of defective proteins through the ubiquitin-proteasome system and the autophagy-lysosome pathway.
Additionally, the maintenance of protein homeostasis is facilitated by the active engagement of ribosome-associated quality control through ribosome pausing (
Brandman et al., 2012;
Howard and Frost, 2021;
Shao et al., 2015). When ribosomes encounter stalling during the translation, ZNF598 identifies the stalled ribosomes and facilitates the activation of eIF2α (
Darnell et al., 2018;
Shao et al., 2015). Consequently, a cascade of ribosomal proteins dissociation factors is recruited to facilitate the degradation of truncated peptides and prevent the aggregation of incorrect peptide segments. This process effectively preserves cellular protein homeostasis. Ribosome pausing is associated with various factors including the cell cycle (
VanInsberghe et al., 2021), leucine limitation (
Darnell et al., 2018), and aging (
Stein et al., 2022). Previous studies have reported that ribosomes tend to pause at proline, glycine, and aspartic acid codon motifs (
Han et al., 2020), with some of these motifs being enriched in guanine codons. In prokaryotes, ribosomes stall at Shine–Dalgarno sequences (
Li et al., 2012), which are composed of adenine and guanine bases. This observation suggests that ribosomes may pause at sequences enriched in guanine. However, the precise underlying mechanism by which aging induces ribosome stalling remains unclear.
G-quadruplexes represent non-canonical secondary structures of DNA or RNA that primarily emerge in guanine-rich regions, displaying diverse topological configurations, including parallel, anti-parallel, and hybrid conformations (
Yuan et al., 2020). Although the structure and function of DNA G-quadruplexes have been comprehensively studied, our understanding of RNA G-quadruplexes (rG4) and their functions remains relatively limited. Putative rG4s are ubiquitously present in both coding and non-coding regions of pre-mRNAs and mRNAs, including introns, 5′- and 3′-UTRs (
Fay et al., 2017;
Kharel et al., 2023). Previous studies have demonstrated that rG4 located in the 5′UTR can impede protein translation, whereas those present in ribosomal genes can suppress protein expression levels (
Murat et al., 2018). However, recent research has shown that 5′UTR rG4s can enhance translation in
Escherichia coli (
Lee et al., 2024). The prevalence of these G4s in mRNA regions associated with regulatory functions suggests their involvement in the regulation of mRNA metabolism.
Here, we provide evidence that ribosome pausing is prevalent in the G-rich regions of mRNA, particularly in senescent cells with higher levels of rG4. Furthermore, our findings demonstrate that the increased localization of rG4 in mRNA leads to enhanced ribosome pausing, which in turn contributes to the disruption of proteostasis and the progression of cellular senescence. Additionally, DHX9 acts as a critical regulator of rG4 abundance, whereby its diminished expression in senescent cells exacerbates ribosome pausing. Moreover, we also observe a similar regulatory mechanism in mouse TFB (tail-tip fibroblast) cells and highly aged tissues of older mice, thereby suggesting a broader regulatory framework. Consequently, our findings reveal that rG4 and DHX9 serve as novel regulators of translation and proteostasis that constitutes a critical mechanism underlying cellular senescence and aging.
Results
Ribosome pausing reduces translation efficiency and is enriched in G-rich regions of mRNA in senescent cells
The collapse of proteostasis is a significant characteristic of cellular senescence (
Lopez-Otin et al., 2013). To elucidate the underlying physiological processes contributing to this phenomenon, we conducted RNA-seq and Ribo-seq analyses on both young (proliferative stage) and replicative senescent BJ fibroblast cells (Figs. 1A and S1A–E). Consistent with previous observations (
Stein et al., 2022), we identified a marked reduction in translation initiation in senescent cells (Fig. S1F). We further investigated the alterations in mRNA translation efficiency (TE) between young and senescent cells. TE is defined as the ratio of ribosome footprints (RFPs) to mRNA abundance, as measured by Ribo-seq and RNA-seq, respectively. We calculated TE using the Xtail algorithm (
Xiao et al., 2016). Remarkably, the number of genes exhibiting reduced TE is approximately four times greater than the count of upregulated genes (Fig. 1B). To further investigate the potential mechanism contributing to TE dysregulation, we conducted an in-depth analysis of the transcriptome and translatome change patterns of genes with downregulated TE (Figs. 1C and S2A). Our analysis revealed that the greatest number of genes displaying changes is exclusively observed at the translational level, specifically in the “RNA none-RFP down” category (Fig. 1C). Additionally, it is noteworthy that ~50% of the genes showing a reduction in TE overlapped with these alterations (Fig. S2B).
To elucidate the factors contributing to the reduced TE, we calculated a ribosome pause score (RPScore) for each position within a coding sequence (CDS) relative to the entire open reading frame. The cumulative distribution of RPScores across the transcriptome revealed no global senescence-related changes (Fig. 1D). With the exception of methionine, there were negligible variations in the relative ribosome density for other amino acids in the context of senescence (Fig. S1G). This suggests that senescence does not induce a systemic alteration in overall elongation pausing. However, considering the possibility that senescence might induce specific changes in translation elongation, we proceeded to extract sequences spanning 20 nucleotides downstream of the downregulated RPScore sites (Fig. 1E) in senescent cells for subsequent motif enrichment analysis. Our findings revealed that the ribosome tended to pause before guanine-rich motifs (Figs. 1F and S2C). When analyzed with rG4 detector, an RNA G-quadruplex predictor (
Turner et al., 2022), the sequences spanning 20 nucleotides downstream of the downregulated RPScore sites exhibited significantly higher RSR-ratio scores compared with shuffled sequences (Fig. 1G). Moreover, ~43.4% (112 out of 258) of the genes displaying translational downregulation also exhibited a concurrent downregulation of RPScore (Fig. 1H). Additionally, we discovered that
FURIN, one of the key endoprotease, exhibited decreased TE in senescent cells and contained regions enriched with guanine nucleotides (Fig. 1I). Overall, ribosome pausing diminishes TE within G-rich regions in senescent cells.
RNA G-quadruplex signal is increased in senescent cells and hinders translation
G-quadruplexes are prone to form, we propose that they influence translation in senescent cells. To test this hypothesis, we conducted experiments to empirically investigate their effects. Following this, we investigated the prevalence of rG4 in senescent cells. In young cells, the G-quadruplex binding protein G4P (
Zheng et al., 2020) and BG4 (
Lee et al., 2024) were predominantly localized within the nucleus. However, in both replication-induced and damage-induced senescent cells, the majority of the G4P and BG4 foci were observed in the cytoplasm, indicating an increased presence of rG4 in senescent cells (Figs. 2A, 2B, and S3A–G). Given that G4P and BG4 are not exclusively specific to rG4, we employed a rG4-specific probe, QUMA-1 (
Chen et al., 2018), for confirmation. Consistently, rG4 levels were found to be significantly increased in both replication- and damage-induced senescent cells (Figs. 2C, S3H and S3I). These findings provide evidence that rG4 levels are elevated in senescent cells.
Next, we established a stable BJ fibroblast cell line expressing the G-quadruplex binding protein G4P and collected both young and senescent cells for RIP-seq analysis (Fig. S4A–C). By integrating these data with the Ribo-seq data, we found that 60.68% of the genes in the overlapping portion between Ribo-seq and G4P-RIP-seq exhibited decreased TE in senescent cells. In contrast, this percentage fell to 48.58% in the non-overlapping sets (Figs. 2D and S4E). Moreover, several genes showing significant fold changes and enrichment in G4P senescent cells also exhibited decreased TE, including RNF213 and HELZ2, both of which contain potential rG4 sequence in their CDS region (Fig. 2E–G). Furthermore, we conducted KEGG and GO analyses on the differentially expressed genes identified from young and senescent G4P-RIP-seq and found that the majority of rG4-enriched genes are implicated in ribosome-related biological pathways (Fig. 2H and 2I). This observation highlights the significant role of rG4 in translation.
RNA G-quadruplexs exacerbate ribosome pausing leading to translation impairment
As previously discussed, the utilization of omics sequencing analysis has provided evidence that rG4 hinders the process of translation. To further substantiate this hypothesis at the molecular level, we developed “mCherry-GFP” dual fluorescence reporter system (Fig. 3A). The mCherry-GFP fusion protein was engineered with either a random sequence or a rG4 sequence, neither of which impeded the transcription (Fig. 3B and 3C). If ribosomes encounter a pause at the rG4 sequence, it would lead to the formation of mCherry-positive but GFP-negative puncta. Subsequently, the reporter system was transfected into BJ cells, resulting in a higher abundance of mCherry
+/GFP
− puncta in the rG4 group compared with the random group. Significantly, the phenomenon observed in the rG4 group was notably augmented by the application of cPDS, a compound known for stabilizing rG4 structures in cells (
Kharel et al., 2023) (Fig. 3D–E). In addition to engineering the artificial rG4 sequence into the “mCherry-GFP” dual fluorescence reporter system, we also inserted actual G4 and rG4 sequences into the reporter system as the sense strand, which are identified from promoter or CDS regions and allow transcription of the “mCherry-rG4-GFP” mRNA. Consistent with the results for the artificial rG4 sequences, all actual rG4 sequences also hindered ribosome elongation, leading to the production of truncated mCherry proteins (Fig. S5A and S5B).
Moreover, we confirmed the inhibition of translation by rG4 through
in vitro cell-free protein synthesis (CFS). We observed a sustained increase in mCherry protein production specifically within the rG4 group, indicating a pause in translation prior to GFP synthesis (Fig. 3F). To substantiate this finding, we performed western blot analysis, which confirmed an increased production of truncated mCherry protein in the rG4 group (Fig. 3G). Ribosome pausing can trigger ribosome collision and activate eIF2α, JNK, and p38 MAPK (
Darnell et al., 2018). Consistent with this, treatment with cPDS led to the phosphorylation of eIF2α (Fig. 3H). To determine whether rG4 sequences contribute to ribosome collisions, we employed cPDS to stabilize rG4 and subsequently conducted polysome profiling. The results showed a significant increase in polysome levels following cPDS treatment (Fig. 3I). Furthermore, in addition to the activation of eIF2α, total translation would be inhibited after cPDS treatment. To validate this assumption, we performed a puromycin incorporation assay, which yielded results consistent with eIF2α activation (Fig. S5C). Moreover, ribosome pausing also contributes to the disruption of proteostasis. Consequently, we examined the aggresome and noted a significant increase in cPDS-treated cells (Fig. 3J–K). These finding provide strong evidence that rG4 exacerbates ribosome pausing.
Stabilization of RNA G-quadruplexs accelerates cellular senescence
Proteostasis imbalance is a key characteristic of cellular senescence. To investigate whether ribosome pausing contribute to the cellular senescence, we employed the “mCherry-GFP” dual fluorescence reporter system and observed a higher abundance of mCherry+/GFP− puncta in the rG4 group in the senescent cells, suggesting that rG4 caused ribosome pausing in senescent cells (Fig. 4A and 4B). Next, in order to prove whether stabilizing rG4 affects the senescence process, presenescent BJ fibroblast cells were subjected to cPDS treatment. SA-β-Gal staining, a widely recognized marker of senescent cells, was performed to assess the effects. The results revealed that cPDS treatment led to a significant increase in the proportion of SA-β-Gal-positive cells (Fig. 4C–F). Additionally, we conducted RNA-seq analyses on both control and cPDS-treated BJ fibroblast cells (Fig. S6) and found that the upregulated genes were notably enriched in cell cycle and cellular senescence processes (Fig. 4G). This suggests that cPDS treatment accelerates cellular senescence. Subsequently, we employed western blot analysis to evaluate the levels of P16, a well-established marker of senescence. It was observed that the levels of p16 increased after cPDS treatment (Fig. 4H).
To further evaluate the senescence process, we treated the cells with etoposide (VP-16) to induce cellular senescence (damage-induce senescence) (
Bang et al., 2019) in combination with cPDS treatment and observed consistent outcomes. This was evidenced by an increased presence of rG4 signals and SA-β-Gal-positive cells (Fig. 4I–L), which aligns with the results observed in the presenescent cells. Furthermore, we investigated the aggresome and identified a significant augmentation in senescent cells, indicating the disruption of proteostasis (Fig. 4M and 4N). In addition to proteostasis, the senescence-associated secretory phenotype (SASP) is another hallmark of cellular senescence, was detected by qPCR which showed a notable increase in the group treated with cPDS (Fig. 4O). These findings provide substantial empirical support for the notion that enhancing the stability of the rG4 structure accelerates cellular senescence.
DHX9 deficiency facilitates RNA G-quadruplex stability and promotes cellular senescence
To elucidate the underlying mechanism responsible for the observed increase in rG4 structures in senescent cells, we conducted a screening of the helicases that bind and unwind rG4 structures by integrating RNA-seq and Ribo-seq analysis. Our findings revealed that only DHX9 and DDX19B exhibited decreased levels of both transcription and translation in senescent cells (Fig. 5A). In addition, our results demonstrated that the deficiency of DHX9 led to a significantly higher increase in the proportion of SA-β-Gal-positive cells (Figs. 5B, 5C, S7A and S7B). This suggests that DHX9 serves as the primary helicase involved in regulating rG4 structures, which also diminished in senescent cells (Fig. 5D). Subsequently, QUMA-1 was performed to detect rG4 levels in DHX9-deficient cells, revealing a notable increase in the abundance of rG4 structures (Fig. 5E and 5F). Moreover, we conducted simultaneous transfection of the “mCherry-GFP” dual fluorescence reporter system and DHX9 siRNA to explore the potential role of DHX9 in regulating translation. The results demonstrated that DHX9 deficiency had no impact on the random group but resulted in an increased number of mCherry+/GFP− puncta and truncated mCherry protein in the rG4 group (Figs. 5G, 5H, S7C and S7D). This suggests an increased frequency of ribosome pausing in DHX9-deficient cells. Conversely, overexpression of DHX9 attenuated the production of truncated mCherry protein and the proportion of SA-β-Gal-positive cells (Figs. 5I and S7E–G). Furthermore, the presence of aggresome was observed in DHX9-deficient cells, with a significant increase observed following DHX9 knockdown (Fig. 5J and 5K). These findings highlight the crucial role of DHX9 in the translation of senescent cells, specifically through its function in the unwinding of rG4 structures.
Disruption of proteostasis accompanied by reduced DHX9 level and increased rG4 abundance in the liver and lung of aged mice
To establish the physiological relevance of this phenomenon, we examined its presence in vivo. The liver, lung, heart, spleen, and kidney were isolated from young mice (2 months old) and aged mice (24 months old) for immunohistochemistry (IHC) analysis (Fig. 6A–H). The results demonstrated a significant reduction in DHX9 expression in liver and lung (Fig. 6B–D), confirming our previous observations in BJ cells. However, DHX9 did not exhibit any noticeable changes in tissues with inherently low DHX9 expression (Fig. 6E–H). Meanwhile, we also assessed the rG4 levels in the tissues and observed a significant increase specifically in the liver and lung of aged mice (Fig. S8). Additionally, we isolated tail-tip fibroblast cells to investigate the level of DHX9, the abundance of rG4, and ribosome pausing (Fig. 6I). The results revealed that tail-tip fibroblast cells from aged mice exhibit a noteworthy reduction in DHX9 (Fig. 6J and 6K) alongside a substantial elevation in rG4 levels (Fig. 6L and 6M). Furthermore, we observed the activation of eIF2α, which led to the inhibition of overall translation in fibroblasts derived from aged mice (Fig. 6N–P). This phenomenon was consistent with the findings in BJ cells. In addition to ribosome pausing, there was an increase in aggresome formation in the fibroblasts of aged mice (Fig. 6Q and 6R), suggesting a disturbance in proteostasis within these cells. Collectively, these observations provide compelling evidence supporting the involvement of DHX9 in the regulation of proteostasis through rG4, both in senescent cells and in aged mice.
Discussion
Ribosome pausing in senescent cells
Ribosome pausing has emerged as a significant focus of research in recent years, primarily attributed to aberrant mRNA, which encompasses RNA modifications and secondary structure (
Yan and Zaher, 2019). Various stress conditions have been reported to result in ribosome pausing and collisions, which may lead to diverse cell fates, such as apoptosis and senescence (
Snieckute et al., 2023;
Stein et al., 2022;
Wu et al., 2020). Currently, investigations into ribosome pausing and cellular senescence predominantly emphasize the role of RNA modifications. Previous studies have indicated that N
6-methyladenosine (m
6A) modifications on mRNA play important roles in translation and cellular senescence (
Boulias and Greer, 2023;
Chen et al., 2022;
Wang et al., 2015). Recently, N
1-methylpseudouridylation of mRNA has been reported to cause +1 ribosomal frameshifting and influence mRNA translation (
Mulroney et al., 2024), suggesting that it may play a role in cellular senescence. In our study, we have identified a novel mechanism of ribosome pausing, wherein the G-quadruplex structure in the mRNA coding region inhibits ribosome elongation, resulting in ribosome stalling and collisions. Furthermore, we observed a significant increase in the abundance of rG4 in the senescent cells, and the stabilization of the rG4 structures by cPDS further exacerbates ribosome pausing and contributes to cellular senescence, as illustrated in Figs. 3 and 4. These findings elucidate the previously unexplored impact of mRNA secondary structures on translation and cellular senescence.
Role of RNA G-quadruplex in mRNA translation
Previous study has demonstrated that rG4 structures located in 5′UTR of mRNA play a crucial role in regulating translation initiation (
Murat et al., 2018). These 5′UTR rG4 structures can recruit the 80S ribosome, thereby inhibiting gene translation. DExH/D helicases, including DHX36, DHX9, and DDX3X, are capable of unwinding rG4 structures located on the 5′UTR or rG4 structures on the 5′UTR of ribosomal proteins, thus facilitating translation process (
Murat et al., 2018;
Varshney et al., 2021). However, existing research primarily focuses on the effects of rG4 structures in the 5′UTR on translation, with limited investigation into the influence of rG4 structures present within the coding regions of mRNA on translation efficacy. Here, we propose that rG4 structures located in the CDS of mRNAs can impede ribosome movement, resulting in ribosome pausing and collision. Stabilization of the rG4 structures by cPDS further exacerbates ribosome pausing.
Furthermore, a significant increase in the abundance of rG4 was observed in senescent cells. Combining this finding with prior research, we postulate that the reduction of helicases during the cellular senescence process could be the underlying cause. To pinpoint the potential helicase, we integrated ribo-seq and RNA-seq data and identified DHX9 as the promising target. Upon validation, it was confirmed that DHX9 is indeed downregulated in senescent cells and aged mice (Figs. 5 and 6). Moreover, deficiency of the RNA helicase DHX9 resulted in an increased presence of rG4 structures, thereby exacerbating ribosome pausing. Conversely, the overexpression of DHX9 alleviated ribosome pausing (Figs. 5 and S6). These findings provide evidence that the presence of rG4 structures within the CDS of mRNAs can induce ribosome pausing and collisions.
RNA G-quadruplex-binding peaks enrich on ribosome genes
There are ~360,000 potential sites for generating G-quadruplex in the genome, but not all genes are capable of forming these structures (
Huppert and Balasubramanian, 2005). To identify the genes whose translation is regulated by rG4, G4P-RIP-seq was conducted to pull down the mRNAs associated with rG4. Subsequent analysis using GO and KEGG revealed that rG4 is enriched on ribosome genes in senescent cells (Fig. 2H and 2I), which is consistent with previous research. This suggests that rG4 initially decreases the TE of ribosomal proteins, potentially impacting ribosome assembly. Furthermore, the occurrence of a cascade reaction results in an insufficient number of functional ribosomes available for the translation of other genes, leading to the collapse of proteostasis that accelerates cellular senescence process.
DHX9 captures mRNA containing rG4 by forming stress granules
DHX9 is the member of the DEAH-box RNA helicase family, which catalyzes the ATP dependent unwinding of RNA or DNA secondary structure. DHX9 plays important roles in many cellular processes, such as DNA replication, cell cycle, and mRNA translation (
Hartman et al., 2006;
Lee et al., 2014;
Zhou et al., 2003). Previous study reported that deficiency of DHX9 induces premature senescence dependent on p53 (
Lee et al., 2014). In this study, we demonstrate a novel mechanism of DHX9 in cellular senescence through unwinding rG4 structures to alleviate ribosome pausing. Recent study has shown that DHX9 can generate stress granules to sequester RNA damaged by ultraviolet light, thereby protecting progeny cells (
Zhou et al., 2024). Additionally, under conditions of starvation or oxidative stress, there is a significant increase in the abundance of rG4 within mRNA (
Kharel et al., 2023). Furthermore, some studies have suggested that rG4 might regulate the formation of stress granules (
Asamitsu et al., 2023;
Danino et al., 2023). Building upon these findings and our own research, it is possible that when rG4 causes ribosomal elongation pausing, DHX9 is likely to recognize and encapsulate these mRNA molecules within stress granules. These stress granules not only provide a more effective solution to ribosomal pausing but also prevent additional ribosomes from binding to the mRNA, thus avoiding new translation challenges. This aspect warrants further exploration and investigation.
DHX9 and RNA G-quadruplex server as the potential targets for delaying cellular senescence
Cellular senescence constitutes a fundamental component of organismal aging, wherein the disruption of proteostasis emerges as a pivotal characteristic. Previous research has predominantly focused on factors such as UPR and autophagy (
Pohl and Dikic, 2019;
Senft and Ronai, 2015;
Sha et al., 2017). Nevertheless, our study uniquely investigates the intricate process of protein synthesis, specifically in translation. Our findings reveal that the deficiency of DHX9 and the stabilization of rG4 both contribute to ribosome pausing, ultimately leading to proteostasis collapse. We propose that in young cells, an adequate presence of DHX9 facilitates the unwinding of rG4 in the mRNA, enabling ribosomes to glide smoothly and efficiently translate proteins. In contrast, senescent cells with a significant reduction of DHX9 exhibit insufficient capacity to unravel the elevated rG4, thereby resulting in ribosome pausing and abnormal protein translation (Fig. 7). Consequently, compounds that activate DHX9 or unwind rG4 hold promise for preserving proteostasis and delaying cellular senescence. Currently, G4 and rG4-related compounds are predominantly employed as stabilizing agents. Nonetheless, there is significant potential in exploring compounds that can unwind rG4, as they may serve as effective therapeutics for delaying cellular senescence.
The Author(s) 2025. Published by Oxford University Press on behalf of Higher Education Press.
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