Regulation and roles of RNA modifications in aging‐related diseases

Abstract With the aging of the global population, accumulating interest is focused on manipulating the fundamental aging‐related signaling pathways to delay the physiological aging process and eventually slow or prevent the appearance or severity of multiple aging‐related diseases. Recently, emerging evidence has shown that RNA modifications, which were historically considered infrastructural features of cellular RNAs, are dynamically regulated across most of the RNA species in cells and thereby critically involved in major biological processes, including cellular senescence and aging. In this review, we summarize the current knowledge about RNA modifications and provide a catalog of RNA modifications on different RNA species, including mRNAs, miRNAs, lncRNA, tRNAs, and rRNAs. Most importantly, we focus on the regulation and roles of these RNA modifications in aging‐related diseases, including neurodegenerative diseases, cardiovascular diseases, cataracts, osteoporosis, and fertility decline. This would be an important step toward a better understanding of fundamental aging mechanisms and thereby facilitating the development of novel diagnostics and therapeutics for aging‐related diseases.


| INTRODUC TI ON
Aging is a natural gradually occurring process of progressive decline in an organism's physiological and psychological adaptability to the environment, culminating in its death. Molecular hallmarks of aging include genomic instability, telomere attrition, epigenetic alterations, loss of proteostasis, deregulated nutrient sensing, mitochondrial dysfunction, cellular senescence, stem cell exhaustion, and altered intercellular communication. In humans, aging can be correlated to increased risks of multiple diseases such as neurodegenerative diseases, cardiovascular diseases, osteoporosis, metabolic dysfunction, defective tissue repair and regeneration, decreased regulation of gut microbes, and cataracts (Cui et al., 2017;Lopez-Otin et al., 2013).
Accumulating studies on aging have revealed that aging phenotypes and aging-related diseases usually result from the complicated interaction between an external environmental stimulus and internal gene expression regulation. Concerning epigenetically regulated gene expression in aging, though most attention has been given to transcriptional alterations, such as DNA methylation patterns, histone modifications, and chromatin remodeling, post-transcriptional regulators, in particular RNA modifications, have been previously underestimated due to the lack of relevant tools to investigate them (Saul & Kosinsky, 2021). One of the first hints in this direction was speculated by a study analyzing the transcriptomes of young and aging mouse livers, which showed that differentially expressed genes in the aging mice liver were significantly enriched in RNA modification-related pathways (White et al., 2015).
RNA modifications play a critical role in nearly every aspect of the biological process, ranging from early embryo development until aging (Mendel et al., 2018). The existence of modified RNA bases was firstly discovered via enzymatic digestion and electrophoresis in the early 1960s and 1970s (Lavi et al., 1977). Thin-layer chromatography, high-performance liquid chromatography (LC), and mass spectrum (MS) were then utilized to determine and detect RNA nucleobase modification based on the differences in the biophysical and biochemical properties between the modified and unmodified bases such as molecular mass, net charge, polarity, and hydrophobicity (Delatte et al., 2016;Jia et al., 2011;Shen et al., 2019). Although these methods are less quantitative, not high-throughput, and may miss the specific RNA context for a modification in some cases, they have expanded the world of RNA modifications. Recently, the next-generation sequencing-based methods with or without using RNA modification specific antibodies, such as m6A-seq, m6Aselective allyl chemical labeling and sequencing (m6A-SAC-seq), deamination adjacent to RNA modification targets (DART-seq), 7-methylguanosine mutational profiling sequencing (m7G-MaP-seq), bisulfite sequencing (BS-seq), and comparative Nanopore direct RNA sequencing, have been developed to probe the specific RNA modification in the whole transcriptome at single-base resolutions (Enroth et al., 2019;Helm & Motorin, 2017;Hu et al., 2022;Leger et al., 2021;Li et al., 2017;Meyer, 2019). Owing to the great advent of methods to detect them qualitatively and quantitatively, hundreds of RNA modifications have now been identified in mammalian cells and all known RNA species, including mRNA, miRNA, tRNA, rRNA, lncRNA, and other non-coding RNAs (Frye et al., 2016), further boosting the epitranscritome studies and suggesting their exigent biological functions. RNA modifications have been reported to critically contribute to nuclear export, translation initiation, transcript stability, splicing, folding, and localization .
Along with the discoveries of RNA modifications, some enzymes that are responsible for writing, reading, and erasing these RNA modifications have also been identified (Kumar & Mohapatra, 2021).
RNA modifications and corresponding modifying enzymes are gaining increasing attention due to their pivotal roles in numerous human diseases, such as obesity, diabetes, neurodegenerative diseases, multiple types of cancer, and even viral infections (Chatterjee et al., 2021;Zhang et al., 2021;Zhou et al., 2020). Many of these linkages arise from mutations and/or single nucleotide polymorphisms (SNPs) in RNA modification-related genes and pathways.
The biochemical aspects of RNA modifications have been extensively reviewed elsewhere (Harcourt et al., 2017). Here, we will introduce the biogenesis and molecular functions of the relatively well-studied RNA modifications in different RNA species. Further, we will focus on the regulation and roles of RNA modifications in aging-related diseases, including neurodegenerative diseases, cardiovascular diseases, cataracts, osteoporosis, and fertility decline.
m6A in the transcriptome at single-base resolution (Hu et al., 2022;Linder et al., 2015), m6A becomes the best-characterized and most abundant internal RNA modification with about 0.2%-0.6% of adenosines having m6A in mammalian mRNAs (Molinie et al., 2016). The transcriptome-wide m6A distribution in mice and humans revealed that m6A is enriched in the coding region and 3′ untranslated regions (3' UTR), with a significant enrichment near the stop codon (Dominissini et al., 2012;Meyer et al., 2012). m6A modification is catalyzed by METTL3-METTL14 complex and their cofactors, such as METTL16, ZCCHC4, RBM15, ZC3H13, VIRMA, CBLL1, and WTAP (Knuckles & Buhler, 2018;Liu et al., 2014;Warda et al., 2017;Wen et al., 2018;Yue et al., 2018). YTHDF1, YTHDF2, IGF2BP1, IGF2BP2, and IGF2BP3 are characterized as reader proteins that recognize the m6A methylation. Two prominent demethylation enzymes are FTO and ALKBH5. Functionally, m6A is involved in almost every step in the mRNA life cycle, from splicing and processing in the nucleus to translation and decay in the cytoplasm (Zhao et al., 2017). At the cellular level, m6A plays a critical role in cellular identity transition between distinct states during differentiation or stress response via influencing the transcriptome output (Zhao et al., 2017). 2.1.2 | N1 methylation of adenosine (m1A) m1A is the methylation of the N1 position of adenosine which was identified in 1961 in tRNA and rRNA (Dunn, 1961). Recently, its presence in eukaryotic mRNA has been demonstrated and its transcriptome-wide distribution has also been mapped via highthroughput methods (Dominissini et al., 2016;Li et al., 2016). m1A is highly enriched around the start codon within the 5'UTR and is preferentially located in highly structured areas (Dominissini et al., 2016;Li et al., 2016). A recent study showed that the presence of m1A blocks reduces RNA base-pairing and induces local RNA duplex melting (Zhou et al., 2016). m1A has been shown to promote translation (Dominissini et al., 2016;Li et al., 2016), although the detailed molecular mechanism is not clear. The only known methyltransferase catalyzing m1A on mRNA is the TRMT6-TRMT61 complex (Safra et al., 2017). It has been reported that YTHDF2 is not only the reader of m6A but also can bind with low affinity to m1A, which suggests its potential role as an m1A reader in cells (Dai et al., 2018).
Moreover, the known erasers of m1A in mRNA are ALKBH1 and ALKBH3 (Aas et al., 2003;Liu et al., 2016). 2.1.3 | 7-Methylguanosine (m7G) m7G is a positively charged RNA modification that is modified by the addition of the 7-methylguanosine "cap" added to the first transcribed nucleotide, which is necessary for the translation of the majority of mRNAs (Cowling, 2009). RNMT is the first identified cap methyltransferase catalyse (Trotman et al., 2017). The reader and eraser proteins are yet to be identified.

F I G U R E 1
Chemical modifications in mRNA 2.1.4 | 2'O-methylation (2'-OMe) 2'-OMe is one of the classical RNA modifications wherein 2′ hydroxyl (-OH) groups have been added to the ribose. The 2'-OMe modification was first demonstrated in bacterial mRNA to affect translation efficiency (Hoernes et al., 2016). 2'-OMe can be added on the N1 (first transcribed nucleotide, m7GpppNmN-) and also on the N2 (second transcribed nucleotide, m7GpppNmNm-), respectively. CMTR1 may act as 2'-O-methyltransferase that modifies the N1 of the mRNA cap (Belanger et al., 2010). There is a need to develop highly sensitive and quantitative methods to gather more information regarding this modification.
2.1.5 | 5-Methylcytosine (m5C) m5C is the most common modification of mRNA where methylation occurs at the 5th position of cytosine and was first reported in 1975.
This modification is similar to DNA methylation m5C except for the ribose. NSUN2 is associated with this mRNA modification. The m5C modification could also be recognized by the mRNA export adaptor protein ALYREFRNA (Bohnsack et al., 2019;Yang et al., 2017), while the exact methyltransferase(s) responsible for m5C modifications in mRNAs is yet to be identified.

| Pseudouridine
Pseudouridine, or isomerization of uridine, also known as 5-ribosyl uracil and Ψ, was first discovered in 1957. This was initially identified as the fifth base in RNA due to its high abundance in cellular RNA. This modification in mRNA is partially attributed to several tRNA and rRNA pseudouridine synthases (PUS) conserved across eukaryotes (Eyler et al., 2019). Further biochemical research and understanding of Ψ in mRNAs are required. Due to its low abundance in mRNA, this modification has not been studied properly until recent technological advances such as the establishment of PseudoU-seq. This results from the post-transcriptional isomerization reaction of uridine (1-ribosyl uracil), which makes pseudouridine carry distinct chemical and biophysical properties compared with uridine. This rigidifies both single-stranded and duplex RNA locally, and thus restricts their flexibility. Expectedly, pseudouridine can affect the secondary structure of mRNA. This modification can have a substantial impact on the translation process and the outcome of translation especially when it occurs in the stop codons or nonsense codons, given that all stop codons start with U at the first base. Ψ-containing codons have been shown to be able to modestly affect the ribosomes, incorporating certain amino acids and Ψ-containing stop codons that have been observed to direct the nonsense suppression of translation termination (Eyler et al., 2019;Fernandez et al., 2013;Karijolich & Yu, 2011). 2.1.7 | Adenosine to inosine RNA editing (A-to-I editing) Like RNA modification, A-to-I editing is a common event in the transcriptome. This conversion is catalyzed by adenosine deaminases acting on the RNA (ADAR) family of enzymes on both intermolecular and intramolecular double-stranded RNAs longer than 20 bp.
Mammals have 3 ADAR enzymes, ADAR1 and ADAR2 being catalytically active while ADAR3 lacks catalytic activity. Considering both coding and non-coding transcripts, tens of thousands of A-to-I editing sites have been identified in mice and millions have been identified in humans. A-to-I editing levels vary across transcripts, tissues, and throughout development ranging from 1 to 100 percent at any given site (Porath et al., 2017;Tan et al., 2017).  (Brummer et al., 2017;Nishikura, 2016). This modification is shown to have a significant contribution and relevance to neural develop-

| RNA modifications on non-coding RNAs
In the last decades, after the discovery of the first noninfrastructural non-coding RNA molecules lin-4 in 1993 (Lee et al., 1993), an explosion of studies has suggested the critical functions of non-coding them for recognition and processing by DGCR8, promoting miRNA maturation. Another methylation modification, "internal m7G," catalyzed by METTL1, has been found to occur on pri-miRNA and affects precursor-miRNA processing. Notably, precursor-miRNA is a double-stranded RNA specie, making them potential substrates for adenosine deaminases acting on RNA (ADAR) enzymes. As miRNA's function is dependent on their base pairing with the target mRNA, A-to-I editing in miRNAs may modulate their target specificity, resulting in decreased suppressing efficiency of one or more downstream target genes. Other modifications such as m5C and pseudouridine can also affect the binding of miRNAs to targets (De Paolis et al., 2021;Han et al., 2021;Zhang et al., 2016).

| Modifications on lncRNAs
Long non-coding RNAs or lncRNA are defined as transcripts longer than 200 nucleotides and not translated into functional proteins.
Various functions and the importance of lncRNAs are getting recognized with the advancement of techniques such as next generation sequencing and exponential growth in our understanding of the genome. Depending upon the cellular localization and specific interactions with DNA, RNA, and proteins, lncRNAs can change the stability and translation of cytoplasmic mRNAs, modulate chromatin function, regulate and/or be involved in the assembly of certain complexes, interfere with signaling pathways (Statello et al., 2021). In contrast, their chemical modifications have not been well explored.
MALAT-1, XIST, and HOX are some of the relatively well-studied examples of RNA modifications in lncRNAs. High-throughput methods revealed that in humans both m6A and m5C were mapped to lncRNAs (Squires et al., 2012). WTAP and METTL16, components of the m6A "writer" complexes can interact with certain lncRNAs whereas methyltransferases of the m5C modification in lncRNAs are not very clear (Dinescu et al., 2019). The tRNA m5C methyltransferase NSUN2 has been identified as the writer responsible for m5C methylation in several lncRNAs (Hussain et al., 2013;Khoddami & Cairns, 2013). Another methylation modification is the m1A, despite its proof of existence in lncRNAs, the specific writers are yet to be identified. Apart from methylation, ψ also occurs in lncRNA and is also catalyzed by PUS.

| Modifications on tRNAs
Transfer RNA species have a typical and distinctive cloverleaf secondary structure (Holley et al., 1965). Unlike mRNAs, tRNAs do not encode proteins but are the direct decoder of codons in mRNAs. They are the connecting link between coding information in nucleotides and amino acids in translated proteins. They are the most heavily modified RNA molecules in terms of quantity and diversity. About 1 out of 5 nucleotides are modified in mammalian tRNAs. Recent studies have shown the importance of RNA modifications at certain positions for tRNA function in key developmental processes. Notably, in animal cells, there are two sets of tRNAs, cytoplasmic tRNAs transcribed from the nuclear genome and the mitochondrial tRNAs transcribed from the mitochondrial genome. In some cases, modifications on different sets of tRNAs are carried out by different enzymes. On the contrary, the same type of modifications on different tRNA species will have different downstream targets and biological effects.

| RNA MOD IFI C ATI ON S IN AG ING -REL ATED NEURODEG ENER ATIVE DIS E A S E S
Aging-related neurodegenerative diseases include Alzheimer's disease (AD), Parkinson's disease (PD), amyotrophic lateral sclerosis (ALS), stroke, and frontotemporal degeneration. Accumulating evidence has indicated that age-related neurodegenerative diseases result from various reasons. Among them, epigenetic changes especially RNA modification that could have serious implications in this aspect should be further explored. m6A is an abundant RNA modification in the brain, and recent studies have demonstrated that the m6A methylation of RNA could promote the development of AD. By using m6A-sequencing together with high-throughput liquid chromatography-tandem mass spectrometry (LC-MS/MS), it is found that the expression level of METTL3 was significantly reduced along with m6A levels in 5xFAD mice when compared with control mice (Shafik et al., 2021). Consistently, the significantly decreased neuronal m6A levels and METTL3 expression were also observed by immunoblot analysis in human AD brains compared with the age-matched control cases . Knockdown of METTL3 in the mouse hippocampus caused memory loss, neurodegeneration, spine loss, and gliosis . Mechanistically, METTL3 deficiency delays the mRNA degradation of m6A-modified cell cycle genes, including Cyclin D1 and Cyclin D2, in the hippocampus and in primary neuron cultures, which causes dysregulated cell cycle and oxidative stress . In addition, a recent study examining the expression profiles of m6A-regulated genes in human AD post-mortem brains has reported the aberrant expression of METTL3 and RBM15B in the AD hippocampus and indicated that the accumulation of METTL3 in the insoluble fractions positively correlated with that of Tau in hippocampal lysates, suggesting that potential perturbations in m6A signaling may contribute towards neuronal dysfunction in AD . Diabetes and obesity are thought to be closely related to AD. It is found that FTO activates mTOR signaling and reduces the mRNA level of TSC1, therefore activating the phosphorylation of Tau in insulin defects-associated AD, and conditional knockout of FTO in the neurons reduces the cognitive deficits in 3xTg AD mice . Besides, the NIA-LOAD study identified a genetic variant in the FTO gene loci significantly associated with AD, and FTO expression was significantly lower in the cortex and amygdala tissues of AD patients compared with controls, suggesting the functional role of FTO in Alzheimer's Disease (Reitz et al., 2012). This is further confirmed by a prospective study showing FTO AA-genotype posed a higher risk for AD and dementia (Keller et al., 2011). These analysis (Zhang, Trebak, et al., 2020). By using microfluidic-based highthroughput PCR along with next-generation sequencing, it was found that A-to-I RNA editing levels were reduced in Alzheimer's disease samples when compared with controls (Khermesh et al., 2016). Similarly, the A-to-I RNA editing events of AD were systematically annotated . 1,676,363 editing sites were detected in 1524 samples across 9 brain regions from ROSMAP, MayoRNAseq, and MSBB studies, within which 108,010 and 26,168 editing events were identified to promote or inhibit AD progression, respectively . 5582 brain region-specific editing events with potentially dual roles in AD across different brain regions were also noticed .

| ALS and RNA modification
ALS is one of the most common age-related neurodegenerative diseases characterized by progressive weakness and muscle atrophy, causing damage to upper and lower motor neurons (Oskarsson et al., 2018). The pathogenesis of amyotrophic lateral sclerosis involves several mechanisms, among which RNA modifications deserve a more thorough investigation.
ADAR2 specifically catalyzes A-to-I RNA editing at the glutamine/ arginine (Q/R) site of GluA2, a subunit in the majority of AMPA receptors in the adult brain, and changes the glutamine (position 607; encoded by CAG) to an arginine (edited to CIG and translated as CGG) within the ion pore of GluA2, which is indispensable for normal AMPA receptor function (Sommer et al., 1991). In conditional ADAR2 knockout mice, the Q/R site of GluA2 cannot be edited by ADAR2, which results in the slow death of the motor neurons Hideyama & Kwak, 2011). Recent evidence demonstrated that the efficiency of RNA editing at the GluA2 Q/R site was significantly lower in all ALS cases compared with that of the control subjects.
Interestingly, of the three members of the ADAR family, only the enzymatic activity of ADAR2 was downregulated in ALS motor neurons, which suggests that once ADAR2 expression levels decreased below the required threshold to edit all GluA2 Q/R sites, motor neurons enter a death cascade. It is also suggested that the progressive downregulation of ADAR2 may be closely related to the pathogenesis of ALS, wherein the failure of A-to-I transition at the GluA2 Q/R locus is critical (Hideyama et al., 2012). Furthermore, the CYFIP2 mRNA K/E site was predominantly edited by ADAR2 and has been newly identified as ADAR-mediated A-to-I editing positions, which could provide a clue to the pathogenesis of ALS (Kwak et al., 2008). Notably, a deeper understanding of the various RNA modifications may shine light on the mechanism-based therapeutic approach to treating age-related neurodegenerative diseases. Similarly, glutamate receptor GRIA2 editing is significantly reduced in the motor neurons of ALS patients as well as in patients with schizophrenia and bipolar disorder. Reduced editing is accompanied by reduced ADAR2 expression in these patients, with RNA editing deficiency contributing to motor neuron toxicity in ALS (Maas et al., 2006). Moreover, TDP-43, a pathological hallmark of ALS, is exclusively expressed in motor neurons lacking ADAR2 in patients with sporadic ALS (Aizawa et al., 2010), demonstrating the pathogenic role of unedited GluA2 at the Q/R locus in ALS.

| RNA MOD IFI C ATI ON S IN AG ING -REL ATED C ARD I OVA SCUL AR D IS E A S E S (C VDS)
CVDs remain the leading cause of death globally (Mortality, & Causes of Death, C, 2013), and biological aging is a major risk factor in CVDs, such as atherosclerosis, coronary heart disease, myocardial infarction, hypertension, stroke, cardiac hypertrophy, and heart failure (HF) (North & Sinclair, 2012). By 2030, approximately 20% of the population will be 65 years of age or older. By that time, CVDs will be responsible for 40% of deaths and the cost of treating CVDs will have tripled (Fleg et al., 2011;Heidenreich et al., 2011). Therefore, it is critical to understand the underlying mechanism by which aging acts as an important determinant of the etiology of CVDs. Here, we discuss some of the associations between RNA modifications and age-related CVDs.

| Atherosclerosis
Atherosclerosis is a chronic inflammatory disease that progresses slowly with the accumulation of cholesterol, lipids, and cellular debris in blood vessels, which in turn greatly increases the risk of restricting blood flow and rupture of blood vessels, contributing to the development of heart attack (myocardial infarction) and stroke (Gistera & Hansson, 2017    . Similarly, cardiac-specific targets of miR-133a were enriched in m6A modifications. IGF2BP2, a key m6A reader, was observed to promote the assembly of the m6A-modified miR-133a-AGO2-RISC complex on the mRNA targets of miR-133a, thereby enhancing the inhibitory effect of miR-133a and protecting from cardiac hypertrophy (Qian et al., 2021).

| RNA modification and other CVDs
Hypertension, one of the most important risk factors for CVDs, is usually defined as a prolonged increase in systemic arterial pressure above a certain threshold (Giles et al., 2009). The incidence of hypertension rises dramatically with age and 70% of older adults have hypertension in 2015 (Mozaffarian et al., 2015). Interestingly, 33 (2.67%) m6A-associated single-nucleotide polymorphisms (m6A-SNPs) were found to be significantly associated with blood pressure in three genome-wide association studies from East Asian populations, within which rs56001051 (C1orf167) and rs197922 (GOSR2) were significantly associated with hypertension (Mo et al., 2019a).
Another study genotyped 217 individuals (86 men and 131 women) with hypertension and found FTO rs9939609 had a negative association with blood pressure in male hypertensive patients (Marcadenti et al., 2013).
The prevalence of HF, characterized as reduced cardiac function and left ventricular dilatation, is predicted to increase remarkably by 46% in the United States from 2012 to 2030 due to the aging population (Heidenreich et al., 2013). Recently, emerging studies have revealed the regulatory mechanisms of RNA methylation in the pathogenesis of HF (Komal et al., 2021). m6A-sequencing and transcriptome analysis of the heart tissues from human HF patients and mouse transverse aortic constriction (TAC) models revealed that m6A modification profiles were changed more dramatically than gene expression and RNAs with altered m6A modification were mainly enriched in metabolic and regulatory pathways (Berulava et al., 2020). Furthermore, cardiomyocyte-specific deletion of FTO accelerated the progression of HF after TAC surgery (Berulava et al., 2020). Cardiomyocyte-specific deletion of ADAR1 leads to an excessive amount of cardiomyocyte loss, resulting in cardiac dysfunction and eventual lethality. Lack of ADAR1 leads to a global reduction in miRNA production, in particular of miR-199a-5p, and the activation of unfolded protein response (UPR)-driven apoptotic response, which hampers ER stress handling in cardiomyocytes.
Inhibition of the UPR in ADAR1-knockout hearts significantly reduced cardiomyocyte loss and restored survival of the animals due to improved cardiac function, which pointed to an essential role for ADAR1 in cardiomyocyte survival and maintenance of cardiac function (El Azzouzi et al., 2020). Interestingly, it is also found that HF patients with reduced ejection fraction had higher concentrations of pseudouridine in plasma compared with healthy controls (Alexander et al., 2011). These studies suggest that RNA modification may serve as therapeutical target against HF.
Stroke is the second most common cause of CVD-related mortality and the number of strokes and deaths due to stroke increases substantially each year (Collaborators, 2021). were significantly hypermethylated after stroke, which may due to the downregulation of FTO (Chokkalla et al., 2019). The integrative analysis of the association between m6A-SNPs and ischemic stroke found that 310 (7.39%) m6A-SNPs were nominally associated with ischemic stroke (Mo et al., 2019b). In addition, another study revealed that YTHDC1 expression was upregulated in the early phase of ischemic stroke, and overexpression of YTHDC1 significantly decreased brain infarct volume . Mechanistically, YTHDC1 activated Akt phosphorylation via promoting the degradation of m6A-modified PTEN mRNA .
Taken together, these studies suggested that epigenetic modifications of mRNAs have a great impact on aging-related CVDs which could provide critical clues to developing future therapies against age-related CVDs.

| RNA MOD IFI C ATI ON S IN AG ING -R E L ATE D C ATA R AC T S
Aging-related cataracts are the main disease of visual impairment and blindness in the world, which commonly occur in people over 50 years old. Cataracts are formed due to the decrease in transparency of the lens , and so far, the only available treatment for cataracts is surgery (Dubois & Bastawrous, 2017).
Therefore, research into the mechanism of cataracts is urgently required to find potential targets for novel therapeutics. A recent study investigated the involvement of m6A circRNAs and methyltransferases in the lens epithelium cells(LECs). By performing genome-wide profiling of m6A-modified circRNAs in lens epithelium cells, they found 2472 m6A peak distributions on 1248 circRNAs with the up-methylation degree and 2174 m6A peaks distribution on 1148 circRNAs with the down-methylation degree. Moreover, the expression of m6A-modified circRNAs in the age-related cataract LECs was lower than that of the controls, which strengthened the dynamic relationship between the m6A modifications at the circRNAs and expression of m6A circRNAs in age-related cataract LECs. They also examined the expression levels of a key methyltransferase, ALKBH5, and two major methyltransferases, METTL3 and WTAP. It was found that the mRNA expression levels of ALKBH5 were significantly upregulated when compared with the control groups, suggesting that ALKBH5 decreases the m6A modifications of circRNAs .
Interestingly, METTL3 could modulate the proliferation and apoptosis of LECs in diabetic cataracts by targeting the 3'UTR of ICAM-1 and stabilizing its mRNA stability .
Isomerization of uridine to pseudouridine is one of the most abundant RNA modifications and is catalyzed by the H/ACA small ribonucleoprotein complex that is composed of four core proteins, dyskerin (DKC1), NOP10, NHP2, and GAR1. Histological analysis has revealed that in Dkc1 elu1/elu1 mutant larvae show microphthalmia and cataracts with abnormal eyes and retinas, accompanied by a large number of cells with neuroepithelial properties (Balogh et al., 2020). Using MeRIP-seq and RNA-seq, one recent study comprehensively analyzed the transcriptome-wide m6A methylome and gene expressions of the anterior capsule of the lens in highly myopic patients with nuclear cataract anterior. They found that METTL14 was upregulated whereas METTL3, FTO, ALKBH5, YTHDF1, and YTHDF2 were downregulated, which suggests that m6A methylation was strongly associated with the pathogenic mechanism of high myopia . Overall, these studies have uncovered the regulatory roles of m6A modifications in aging-related cataracts.

| RNA MOD IFI C ATI ON S IN AG ING -REL ATED OS TEOP OROS IS
Aging-related osteoporosis is characterized by low bone mass and over-accumulation of fatty tissue in the bone marrow environment that increases the risk of fracture (Duque et al., 2009). With aging, the composition of the bone marrow shifts to favor the presence of adipocytes, osteoclast activity increases and osteoblast function declines, leading to osteoporosis (Coughlan & Dockery, 2014). METTL3, the key methyltransferase of m6A, was observed to regulate the fate of osteoporosis. Firstly, the expression levels of METTL3 and m6A methylation are significantly decreased in both osteoporosis patients and mouse models . Downregulation of METTL3 caused the decline in bone formation and overexpressed METTL3 could partially rescue the feature of osteoporosis such as reduction in bone formation. Molecularly, METTL3 mediates m6A methylation of RUNX2, a key factor involved in osteogenesis, and enhances its cellular stability . METTL3 knockout reduced the translation efficiency of MSCs lineage allocator Pth1r and then led to a reduction of the global methylation level of m6A and disruption of the PTH-induced osteogenic and adipogenic responses, which eventually affects the osteogenic and adipogenic differentiation of mesenchymal stem cells (Wu et al., 2018). Apart from this, a recent study revealed different molecular mechanisms of METTL3-dependent m6A modification in osteoclast differentiation.
Here, the depletion of either METTL3 or YTHDF2 promoted the stability and the expression of Atp6v0d2 mRNA . Here, miR-103-3p inhibits osteoblast activity by directly targeting METTL14 while METTL14-dependent m6A methylation enhances the recognition of pri-miR-103-3p by DGCR8 and the subsequent processing into mature miR-103-3p, thereby modulating osteoblast activity .
FTO is a key regulator associated with adipogenesis, and the complete depletion of FTO in mice results in postnatal growth retardation. FTO knockout mice have not only a significantly shorter body length over the lifetime but also a much lower bone mineral density (Gao et al., 2010). To evaluate the effect of FTO on bone mass and to prevent the potential confounding effect of FTO on global metabolism and body composition, mice lacking FTO selectively in osteoblasts (FTO Oc KO ) were generated. These mice showed a significant decrease in bone volume and trabecular number at 30 weeks of age.
Furthermore, the results of static and dynamic histomorphometric analyses showed that the bone formation rate in mutant mice was decreased by 66% with bone marrow adipocyte number per bone marrow area being increased when compared with controls. FTO functioned through demethylating and then enhancing the stability of the mRNAs of Hspa1a and other genes that can protect cells from genotoxic damage . The above results implied that FTO is required for the maintenance of bone mass and FTO Oc KO mice manifest the phenotype consistent with age-related bone loss. miR-149-3p was found to directly target FTO mRNA and modulate the adipogenic differentiation of bone marrow-derived mesenchymal stem cells . Besides, during aging and osteoporosis, FTO was upregulated by GDF11 in both humans and mice, and then stabilized the Pparg mRNA through the demethylation of m6A, leading to the differentiation of bone mesenchymal stem cells to adipocytes rather than osteoblasts .
FTO also plays an intrinsic role in osteoblasts by enhancing the stability of mRNAs of proteins that can protect cells from genotoxic damage via Hspa1a-NF-κB signaling . Altogether, these findings provide new perspectives on the pivotal role of m6A in regulating age-related bone diseases such as osteoporosis.

| RNA MOD IFI C ATI ON S IN AG ING -REL ATED FERTILIT Y DECLINE
Age-related fertility decline is inevitable and irreversible, especially for female reproductive potential. Several demographic and epidemiological studies have long recognized that female fertility declines with age, most notably the decline in ovarian function (Leridon, 2004;Menken et al., 1986;Nelson et al., 2013). The mechanisms involved in the process of ovarian aging have gained increased attention and focus. Herein, we highlight the importance of RNA modification in ovarian aging.
All m6A modifications of the granulosa cells of aged human ovaries were measured in order to investigate the relationship between ovarian aging and m6A modification. It was found the level of m6A modifications was significantly increased and the expression level of FTO was downregulated. m6A sequencing showed that increased m6A in the 3′UTR of FOS mRNAs resulted in reinforcing the stability of FOS mRNAs . Another study also concurrently found that the expression of FTO decreased and the content of m6A increased with aging in human follicular fluid, granulosa cells, and mouse ovary . Besides, the chemotherapy drug, cyclophosphamide, could increase the m6A level and significantly inhibit the expression levels of RNA demethylase FTO in a time-and concentration-dependent manner, which is further associated with premature ovarian aging . More research exploring the precise mechanism of RNA modifications in ovarian aging would give insight into possible strategies to postpone ovarian aging.

| CON CLUS I ON AND PER S PEC TIVE S
In conclusion, the field of epitranscriptomics has emerged rapidly in recent years and RNA modifications have been emerging as a new focus and novel therapeutical targets against aging-related diseases. In this review, we have summarized RNA epitranscriptomic regulation and the mechanisms involved in aging-related diseases.
We found that RNA modification is involved in many diseases that are aging-related, and plays an essential role in impacting mRNA stability, translation, and control of protein levels of key genes that are involved in pertinent disease-associated pathways (Table 1).  (Wu et al., 2018) Osteoclast differentiation m6A Increased METTL3 expression METTL3 deficiency promotes the stability and the expression of Atp6v0d2 mRNA and reduced the expression level of Vegfa and its splice variants Tian et al., 2019) osteoclast differentiation m6A Increased METTL3 expression and decreased ALKBH5 expression Facilitating m6A modifications of MYD88-RNA and then inducing the activation of NF-κB  Osteoblast activity m6A Increased miR-103-3p level and decreased METTL14 expression Regulating the maturation process of miR-103-3p, which directly targets METTL14 to inhibit osteoblast activity  Maintenance of bone mass Increasing the stability of FOS mRNA  TA B L E 1 (Continued)  modification is increasingly being studied in other fields, there are still many modifying erasers/writers/readers that remain to be discovered. Their functions and potential therapeutic implications in aging-associated diseases have yet to be investigated. Moreover, few studies that have focused on m6A application and m6Atargeting drug therapy need to be further explored in-depth.
Although an apparent association of 10 m6A-modification genes and sporadic PD in the Chinese cohort was not found, further functional studies are needed to explore the association between RNA modifications and PD, given the impact of RNA modification on brain development and other aging-related neurological disorders . There are very few studies on RNA modification related to macular degeneration and the regulation of gut microbes, which are areas worth exploring to gain more knowledge and advance the field of aging-associated RNA modifications. Taken together, further efforts are required to gain an in-depth insight into the role of RNA modifications in aging-related diseases and would provide new potential molecular targets for research and development of pharmacological and clinical therapies for many agingrelated diseases. F I G U R E 3 RNA modification genes associated with aging-related diseases

CO N FLI C T O F I NTE R E S T
The authors declare that they have no conflict of interest.

DATA AVA I L A B I L I T Y S TAT E M E N T
Data sharing is not applicable to this article as no new data were created or analyzed in this study.