Caenorhabditis elegans is an excellent model organism for genetic dissection of diverse physiological processes, including aging (Lee and Lee, 2022; Lee et al., 2021). The advantages of C. elegans for the aging research include a relatively short lifespan (2-3 weeks), genetic tractability, and many conserved aging-regulatory pathways shared with mammals. Indeed, diverse genes, pathways, and environmental factors that affect aging and longevity in multiple species have been first identified in C. elegans, and subsequently confirmed in other species (Kenyon, 2010; Lee et al., 2021). Representative evolutionarily conserved longevity-promoting regimens include reduced insulin/insulin-like growth factor 1 (IGF-1) signaling (IIS), dietary restriction (DR), inhibition of mechanistic target of rapamycin (mTOR) signaling, and mild inhibition of mitochondrial respiration (Kenyon, 2010; Lee and Lee, 2022).

Aging is accompanied by the gradual decline in cellular and organismal fitness. At the macromolecular level, impaired protein homeostasis (proteostasis) and genome integrity are key features of aging (Lee et al., 2021; López-Otín et al., 2023; Son et al., 2019). The age-dependent deteriorative changes are interconnected with each other to decrease the lifespan and increase the incidence of age-associated diseases, which eventually lead to death. Similar to other species, C. elegans exhibits age-associated changes such as the reduction in genome stability, proteostasis, lipid homeostasis, and immunity (Jung et al., 2020; Kim et al., 2022; Lee et al., 2019; Lu et al., 2022; Son et al., 2019). Recent studies have indicated that RNA quality also declines during aging in C. elegans.

Eukaryotes are equipped with homeostatic systems that are crucial for the maintenance of mRNA quality, which is regulated by diverse surveillance pathways. Misprocessed mRNAs need to be eliminated by these RNA surveillance pathways. Nonsense-mediated mRNA decay (NMD), no-go decay, nonstop decay, and ribosome-associated quality control (RQC) are crucial for mRNA and protein quality control (Powers et al., 2020). mRNA splicing, which is a major pre-mRNA processing event in eukaryotes, selects and joins exons that are separated by introns, thus enabling diverse gene expression (Rogalska et al., 2023). Changes in splicing occur during aging and may reflect the deteriorated transcriptome quality (Ham et al., 2022; Heintz et al., 2017). Abnormal mRNA splicing underlies the generation of aberrant transcripts that disrupts the proteostasis by producing truncated proteins and causing ribosome stalling, followed by ribosome collision. Here we review recent studies that report on the key functions of various factors that regulate mRNA surveillance and splicing in the longevity and aging of C. elegans. Our review provides crucial information regarding the conserved functions of mRNA quality control in aging, which may be potentially utilized as therapeutic targets of aging and age-associated diseases in humans.

NMD promotes longevity conferred by reduced IIS and DR in C. elegans

NMD is an evolutionarily conserved process that is crucial for cellular and organismal mRNA quality (Behm-Ansmant et al., 2007; Hwang et al., 2021; Kim and Maquat, 2019; Schweingruber et al., 2013). The NMD complex recognizes the aberrant transcripts, such as premature termination codon (PTC)-containing mRNAs, for degradation through the action of nucleases. In C. elegans, the NMD complex is composed of multiple suppressor with morphogenetic effect on genitalia (SMG) proteins, SMG-1 to SMG-7 (Hodgkin et al., 1989; Mango, 2001). The key component of the NMD complex is the SMG-2/up-frameshift 1 (UPF1), which is an ATP-dependent RNA helicase (Hwang et al., 2021; Kim and Maquat, 2019). The activity of SMG-2 is dependent on its phosphorylation by SMG-1 kinase in association with other SMG components.

Recent studies have indicated that NMD-mediated mRNA quality control is crucial for longevity in C. elegans (Fig. 1) (Kim et al., 2020; Park et al., 2017; Son and Lee, 2017; Son et al., 2017; 2019; Tabrez et al., 2017). The activity of NMD declines during aging, as the level of PTC-containing NMD reporters increases in multiple tissues of aged animals (Son et al., 2017). SMG-2/UPF1 is required for long lifespan conferred by various regimens in C. elegans, including daf-2/insulin/IGF-1 receptor mutations, mitochondrial respiration isp-1/Rieske iron-sulfur protein in complex III mutations, germline loss, and DR (Son et al., 2017). In addition to smg-2, smg-1, -3, -4, and -5 are required for the increased lifespan of daf-2 mutants. These data indicate that NMD, whose activity decreases with age, is a biological marker of aging and contributes to the longevity of C. elegans.

Further analysis identified the tissues and target genes that are crucial for NMD-mediated longevity caused by reduced IIS (Son et al., 2017). Neuron-specific smg-2 knockdown substantially decreases the long lifespan of daf-2 mutants, and conversely neuron-specific smg-2 expression restores longevity in the smg-2; daf-2 double mutants (Son et al., 2017). These data suggest that NMD activity in neurons is critical for longevity conferred by reduced IIS. Although smg-2 overexpression has little effect on lifespan, overexpression of smg-1, which encodes the SMG-2-activating protein kinase, increases lifespan (Son et al., 2017). Thus, upregulation of NMD through the activation of SMG-2 extends lifespan. Transcriptome analysis reveals that the levels of endogenous NMD targets, such as PTC-, uORF (upstream open reading frame)-, and long 3′-UTR (untranslated region)-containing transcripts, are reduced in daf-2 mutants compared to wild-type C. elegans. Among the transcripts downregulated in the daf-2 mutants, the mRNA level of yars-2, which encodes a mitochondrial tyrosyl-tRNA synthetase, is decreased in a smg-2-dependent manner. Importantly, the genetic inhibition of yars-2 suppresses short lifespan in smg-2; daf-2 double mutants, indicating that the degradation of an aberrant PTC-containing yars-2 mRNA isoform by NMD contributes to the longevity of daf-2 mutants (Son et al., 2017). Overall, the maintenance of neuronal RNA homeostasis by NMD is essential for the longevity conferred by reduced IIS.

NMD also promotes the longevity conferred by DR in C. elegans (Son et al., 2017; Tabrez et al., 2017). DR-mimetic eat-2 mutants display enhanced NMD activity and require smg-2 for their conferred longevity (Son et al., 2017; Tabrez et al., 2017). The mRNA levels of NMD component genes, smg-1 to smg-7, except for smg-5, are increased in eat-2 mutants (Tabrez et al., 2017). Upregulation of smg-2 in eat-2 mutants depends on the splicing mediator HRPU-1/heterogeneous nuclear ribonucleoprotein U (HNRNPU), suggesting that alternative splicing is also associated with the role of NMD in longevity caused by DR (Tabrez et al., 2017).

After establishing the important role of NMD in aging, a study sought to understand how upstream factors affect the functions of NMD and consequently aging (Kim et al., 2020). Through performing mutagenesis and genome-wide RNAi screens, ALGN-2/alpha-1,3/1,6-mannosyltransferase (ALG2) was identified as a novel positive upstream regulator of NMD, which promotes the longevity of C. elegans (Kim et al., 2020). The mRNA level of algn-2 decreases with age (Kim et al., 2020), consistent with the age-dependent decline in NMD function (Son et al., 2017). ALGN-2 is necessary and sufficient to lengthen lifespan (Kim et al., 2020). The level of ALGN-2 is increased in daf-2 mutants and contributes to the reduced IIS-mediated longevity by enhancing NMD activity in a SMG-2-dependent manner. These findings provide crucial information regarding the role of NMD and its regulators in longevity-promoting signaling pathways. Further investigation into how a mannosyltransferase ALGN-2 regulates NMD during aging will be an exciting future research avenue.

mRNA translation kinetics and RQC are impaired during aging in C. elegans

Damage to various regions in mRNAs and the disruption of the secondary structures cause ribosome stalling, which impairs proteostasis by producing truncated polypeptides (Brandman and Hegde, 2016; Joazeiro, 2019; Park et al., 2021; Yan and Zaher, 2019). RQC regulates the ribosomes and nascent polypeptides to mitigate the detrimental effects of ribosome stalling and collision. RQC is divided into several steps: the recognition and splitting of stalled ribosomes, triggering of RQC, degradation of aberrant nascent polypeptides, and decay of associated mRNAs by exonucleases and endonucleases.

A recent study reported that ribosome stalling increases with age in both C. elegans and Saccharomyces cerevisiae, by using ribosome footprinting (Fig. 2) (Stein et al., 2022). The age-dependent increase of ribosome stalling preferentially occurs in basic amino acids. In addition, aged C. elegans exhibits an increase in ribosome stalling at the polybasic regions. Aging also decreases the RQC activity and increases the level of aberrant polypeptides co-translationally, thereby causing their aggregation in yeast and C. elegans. Genetic depletion of RQC components shortens lifespan in yeast, which suggests that RQC is crucial to maintain a normal lifespan. Thus, it seems likely that aging contributes to the production and accumulation of aberrant peptides, which are generated from impaired RQC. However, the functionally important aging-associated targets of RQC and their roles in metazoan longevity remain elusive. Thus, it is important to identify these factors and to elucidate the underlying molecular mechanisms to understand the relationship between RQC and healthy longevity.

Splicing-mediated mRNA quality control contributes to longevity

DR slows the aging process and increases lifespan in virtually all tested organisms ranging from yeast to mammals (Fontana et al., 2010). In C. elegans, DR causes global changes in the splicing activity that extends the period of youthful splicing patterns, thus contributing to longevity (Heintz et al., 2017). This appears to be achieved through the activation of the quality control mechanisms that counteract the production of deleterious transcripts during aging. As described above, DR also increases alternative splicing-mediated NMD to promote longevity (Tabrez et al., 2017). Thus, DR slows aging and extends lifespan in C. elegans by enhancing the mRNA quality, which prevents harmful splicing events that are associated with old age.

The mTOR signaling pathway is highly conserved across eukaryotic species and regulates the metabolic processes that are crucial for growth, development, and aging (Blackwell et al., 2019; Lee et al., 2015). Genetic inhibition of the mTOR ortholog, let-363, increases lifespan in C. elegans (Vellai et al., 2003). Downregulation of mTOR reduces the overall translation, and subsequently lowers the accumulation of age-associated misfolded proteins, leading to slowed aging and extended lifespan (Lee et al., 2015). mRNA splicing that is regulated by splicing factors such as SFA-1/splicing factor 1 (SF1) and RNP-6/poly-U binding splicing factor 60 (PUF60) plays crucial roles in the C. elegans longevity conferred by reduced mTOR signaling (Heintz et al., 2017; Huang et al., 2022). RNAi knockdown of sfa-1 suppresses the longevity conferred by the reduced mTOR signaling, including mutations in genes that encode key mTOR signaling components, RAGA-1/ras-related GTP-binding A (RRAGA) and RSKS-1/ribosomal protein S6 kinase B2 (RPS6KB2). In addition, reduction-of-function mutations in rnp-6/PUF60 increase the intron retention of egl-8/phospholipase Cβ4 (PLCB4), thereby decreasing the relative level of normally spliced egl-8 mRNA (Huang et al., 2022). This downregulation of egl-8 subsequently decreases the mTOR signaling, leading to an increased lifespan. These findings highlight the combined actions of splicing factors and mTOR signaling that are crucial for longevity.

Changes in the alternative splicing is a prevalent aging feature in multiple species (Bhadra et al., 2020). Some of these age-dependent changes occur chronologically and are independent of age-associated physiological deterioration. Other age-related changes reflect the physiological age that underlies the decline in the youthfulness of organisms. A recent study comprehensively analyzes the transcriptomic changes that are associated with chronological and physiological aging (Fig. 3A) (Ham et al., 2022). Chronological aging is accompanied by an increase in the levels of nonexonic RNAs and noncoding RNAs, as the changes are similar between the aged wild-type C. elegans, and daf-2 mutants, which display dramatically slowed aging rates. In contrast, the usage of distal 3′ splice sites among the changes in transcripts is increased in aged wild-type animals, but this increase is decelerated by daf-2 mutations. Thus, the increased usage of distal over proximal 3′ splice sites is a transcriptomic feature of physiological aging. These changes occur in both the somatic and reproductive tissues, which suggests that increased distal 3′ splice site usage is a ubiquitous age-associated process. Further, the analysis identified transcripts encoding RNA-processing factors that are differentially enriched in daf-2 mutants compared to wild-type animals. Several of these RNA-processing factors, including F30A10.9, the rRNA-processing protein FCF1 ortholog, are required for longevity and the decelerated usage of distal 3′ splice sites in representative transcripts during aging in daf-2 mutants. Thus, RNA-processing events that are regulated by various proteins, including F30A10.9, underlie the transcriptomic changes associated with physiological aging. Future research to determine how these RNA-processing events are impaired during aging will be important to understand the specific molecular mechanisms.

Age-associated changes in the transcription rates can affect the generation of splicing products in C. elegans (Fig. 3B). Transcription elongation by RNA polymerase II increases in speed with age in C. elegans, fruit flies, mice, rats, and humans (Debès et al., 2023). This increase in transcription speed appears to be caused by the dysregulation of transcription elongation in a global manner, regardless of intron position, length, and tissue type. Fast transcription elongation correlates with increased splicing rates, leading to the age-associated upregulation of rare splicing events, mismatched transcript products, and erroneously spliced sequences. Importantly, genetic changes that reduce the transcription elongation speed promote the longevity of C. elegans and Drosophila melanogaster. In addition, daf-2 mutations and DR decrease the transcription elongation rates in old C. elegans. Thus, reducing the transcription elongation speed can delay aging and lengthen lifespan. This work suggests that the mismanagement of transcriptional products and splicing dysregulation facilitates aging in diverse species, including C. elegans.

Studies have identified various factors that may regulate splicing and thereby affect the aging and lifespan of C. elegans. In addition to the RNA helicase SMG-2 that is discussed above, SACY-1/DEAD-box helicase 41 (DDX41) and HEL-1/DExD-box helicase 39A (DDX39A), are two other RNA helicases that are implicated in splicing and contribute to longevity (Park et al., 2017; Seo et al., 2015; 2016). SACY-1 is necessary for lifespan extension caused by multiple longevity-promoting regimens, including reduced IIS, DR, sensory deprivation, germline loss, and mitochondrial impairment (Seo et al., 2016). Based on the functions of its human homolog, SACY-1 is likely to function as a splicing regulator that affects the mRNA quality. HEL-1 mediates the lifespan extension conferred by daf-2 mutations through binding and activating DAF-16/forkhead box O (FOXO) (Seo et al., 2015). Although reduction-of-function mutations in hel-1 cause only slight increases in the levels of tested unspliced mRNAs, perhaps the specific target transcripts of HEL-1, which have not yet been identified, may play a key role in lifespan extension. tcer-1/transcription elongation regulator 1 (TCERG1), which is a transcription elongation and splicing factor (Amrit et al., 2016; Sánchez-Hernández et al., 2016), is required for the increased lifespan conferred by germline removal (Ghazi et al., 2009). Overexpression of tcer-1 increases the lifespan at the cost of compromising various health indicators, such as stress resistance and immunity (Amrit et al., 2019). The muscleblind-like protein 1 (MBL-1) is the C. elegans ortholog of the mammalian muscleblind-like splicing regulator MBNL proteins, which control the mRNA splicing in the muscles and neurons (Charizanis et al., 2012; Kanadia et al., 2003). MBL-1 is required for maintaining a normal lifespan through the regulation of PMK-1/p38 mitogen-activated protein kinase (p38 MAPK) and SKN-1/nuclear factor erythroid 2-related factor (NRF) (Matilainen et al., 2021). The short lifespan of mbl-1 loss-of-function mutants is substantially prolonged through the mild inhibition of mitochondrial respiration. Thus, the proper reduction of mitochondrial respiration, which is a conserved longevity regimen (Haynes and Hekimi, 2022; Hwang et al., 2012), may prevent the detrimental effects of impaired splicing on lifespan. PRP-38/pre-mRNA processing factor 38A (PRPF38A) interacts with an alternative splicing regulator, microfibril-associated protein-1 (MFAP-1) (Ma et al., 2012). The genetic inhibition of prp-38 during adulthood using RNAi substantially increases the lifespan of C. elegans (Curran and Ruvkun, 2007). Although the specific mechanisms are unclear, PRP-38 may affect lifespan as a component of a large spliceosome complex. Future research on these splicing regulators will further elucidate the roles of mRNA splicing processes that affect lifespan and aging in C. elegans.

Impaired cellular homeostasis is a hallmark of aging (López-Otín et al., 2023). The age-dependent impairment of diverse cellular quality control systems that maintain DNA and proteins has been extensively investigated, and the compromised transcriptional fidelity has recently emerged as an integral aspect of aging. In this review, we discussed key recent findings regarding the aging-regulatory roles of RNA quality control, focusing on NMD, RQC, and splicing fidelity, in C. elegans. NMD is essential for the longevity conferred by reduced IIS and DR in C. elegans. RQC activity is decreased with age and is important to maintain a normal lifespan. Proper splicing is essential for the functionality of several proteins that mediate longevity. The studies discussed here highlight the important roles of mRNA quality control acting with diverse biological pathways that affect longevity.

Many important questions regarding the relationship between mRNA quality and aging remain to be answered in future studies. For example, the specific molecular factors that are prone to deterioration of transcriptome quality at the cellular level are not completely understood. This is largely due to the scarcity of reliable methods to produce transcriptomic perturbations and to analyze the ramifications of mRNA dysregulation on cellular homeostasis. Recent advances in single-cell RNA sequencing will be useful for precisely detecting age-associated changes at the single cell level (Junaid et al., 2022; Kim and Kim, 2021; Roux et al., 2023), which may have been masked in bulk RNA sequencing analysis. Aging- and longevity-associated NMD targets are largely unknown, and therefore it is crucial to identify and characterize the specific roles of these factors in NMD-mediated longevity. RQC has been extensively studied using the budding yeast and cultured human cells. However, little is known regarding the co-translational regulatory effects of RQC on aging and healthy longevity. mRNA modification such as N⁶-adenosine methylation (m⁶A), the most abundant RNA modification, modulates mRNA quality by affecting splicing, localization, and stability (Jang et al., 2022). Studies on the assessment of age-dependent changes in m⁶A modification and their roles in aging are limited. Extracellular vesicles (EVs), which contain macromolecules including RNA and mediate cell-cell communications, are secreted from senescent cells for promoting the senescence of recipient cells (Oh et al., 2022). However, it remains unknown whether the quality of RNA components in EVs is changed during aging or plays any role in the longevity of C. elegans. Future studies addressing these issues will be exciting research avenues in the field of aging biology.

Although reports showing direct causal relationship between mRNA quality systems and aging are rare in mammals, many studies suggest the roles of NMD, RQC, and mRNA splicing in age-associated diseases in mammals, including humans. NMD plays both negative and positive roles in age-associated diseases such as Alzheimer’s disease (AD), amyotrophic lateral sclerosis, and cancer in mammals (Barmada et al., 2015; Cao et al., 2017; Jackson et al., 2015; Ju et al., 2011; Wang et al., 2011). Impaired RQC and excessive ribosome stalling are implicated in many neurodegenerative diseases, including AD and Parkinson’s disease (Chu et al., 2009; Giovannone et al., 2009; Ishimura et al., 2014; Martin et al., 2020; Rimal et al., 2021; Wu et al., 2019). In addition, splicing dysregulation is implicated in cellular senescence, and age-related neurogenerative diseases in humans (Angarola and Anczuków, 2021; Deschênes and Chabot, 2017; Ham and Lee, 2020; Holly et al., 2013; Mazin et al., 2013; Tollervey et al., 2011; Wang et al., 2018). Future research utilizing the information obtained from aging and longevity research with C. elegans and from age-associated diseases in mammals will pave ways to understand the functions of RNA quality control systems in healthy aging in humans.

The fundamental goal of aging research is the extension of healthspan, lifespan with healthy periods, in humans. As aging perturbs the homeostasis of RNA as well as DNA and proteins, it is crucial to maintain the homeostasis of all these three macromolecules. The studies covered in this review indicate that the proper maintenance of mRNA quality via enhancing surveillance and splicing systems is necessary and sufficient for healthy longevity in C. elegans. Numerous discoveries in the aging research field that were first identified using C. elegans have been demonstrated to be conserved in mammals. Therefore, investigating the roles of RNA homeostasis in aging and longevity in C. elegans may provide insights into the development of effective strategies for healthy longevity in humans.

We thank all Lee laboratory members for helpful comments and discussion. This research was supported by the KAIST Key Research Institutes Project (Interdisciplinary Research Group) to S.J.V.L.

H.C.K., Y.B., and S.J.V.L. wrote the paper.

The authors have no potential conflicts of interest to disclose.