N6-Methyladenosine Modifications in the Female Reproductive System: Roles in Gonad Development and Diseases.

There is evidence that m 6 A modification takes part in the process of inflammation and immune activity 102 , 103 . Considering that inflammatory or immune disorders play a critical role in many female reproductive diseases such as endometriosis and adenomyosis, polycystic ovary syndrome, preeclampsia and spontaneous abortion 104 , 105 , it stands to reason that m 6 A modification may also contribute to the pathological mechanisms underlying these diseases. Bioinformatic analyses revealed that most of the m 6 A regulators in the eutopic endometrium were aberrantly expressed compared to normal or eutopic endometrium. Among all the regulators, HNRNPA2B1 and HNRNPC were negatively correlated with the severity of endometriosis, and might influence infiltrating immune cells 106 . The m 6 A content was found to be dramatically decreased in eutopic and ectopic samples, which was consistent with decreased METTL3 and increased ALKBH5 levels. METTL3 regulates the migration and invasion of endometrial stromal cells by facilitating DGCR8-mediated maturation of miR126, contributing to endometriosis development 107 . Similarly, the expression pattern of m 6 A regulators in the myometrium was also found to be different from normal tissues. The cluster of m 6 A regulators may potentially affect the immune response and cell adhesion according to data mining, playing roles in the myometrium dysfunction 108 . Polycystic ovary syndrome (PCOS) is often accompanied by insulin resistance. FTO overexpression diminished m 6 A modification in FLOT2 transcripts and enhanced its expression in granulosa cell line KGN. FLOT2 is associated with insulin resistance and was found to be upregulated in granulosa cells from PCOS patients. FTO was found to regulate cell proliferation, apoptosis and insulin resistance in KGN cells through a FLOT2-dependent manner, indicating that FTO might participate in regulating insulin resistance in PCOS 109 . Luteinized granulosa cells from non-obese PCOS patients had higher m 6 A levels and exhibited a dissimilar distribution pattern of m 6 A peaks in comparison to the control group. Mechanistically, the FOXO3 transcript was m 6 A modified and underwent YTHDF2‐mediated decay in controls, which was in contrast to the samples from PCOS patients. Reduced m 6 A modification of the FOXO3 mRNA in controls enhanced the expression of FOXO3 after METTL3 or METTL14 knockdown, while having the inverse effect after FTO knockdown. However, this effect was absent from the PCOS granulosa cells 110 . These findings illustrate a potential mechanism driving insulin resistance in PCOS patients. In addition to increased m 6 A modification in naturally aging mouse ovaries 111 , the levels of m 6 A modification ware also found to be obviously upregulated in either ovarian tissues or human granulosa cells from premature ovarian insufficiency (POI) patients. This elevation was associated with a relative reduction of FTO compared to ALKBH5. FTO knockdown impeded cell proliferation, alleviated apoptosis and disturbed marker expression in human granulosa cells 112 . Furthermore, m 6 A was also increased in a mouse model of cyclophosphamide (CTX)-induced premature ovarian insufficiency in a concentration- and time-dependent manner. CTX treatment upregulated the m 6 A writers METTL3, METTL14, ZC3H13 and KIAA1429, while downregulating the demethylase FTO and several m 6 A readers 112 , 113 .These results indicate that m 6 A modification may act as a potential biomarker of ovarian dysfunction 112 , 113 . Dysfunction of the placenta in preeclampsia and eclampsia patients usually leads to abnormal fetal growth and serious complications for the pregnant women. Previous studies have identified the role of epigenetics in preeclampsia and fetal growth restriction 114 , 115 . The potential involvement of m 6 A modifications in the pathological processes of preeclampsia has been proposed based on several lines of evidence. MeRIP-seq analysis demonstrated a correlation between higher m 6 A levels at the 5′‐UTR in placental mRNAs and small‐for‐date children, whereas decreased m 6 A levels near stop codons were dominant in heavy‐for‐date placenta samples. Higher SMPD1 protein levels in preeclampsia placenta samples, in particular, are more likely to result from m 6 A enrichment at 5′‐UTR instead of increased mRNA levels 116 . Enhanced METTL3 expression was found to be responsible for increased m 6 A methylation in placental trophoblasts from preeclampsia patients. These aberrant m 6 A changes lead to the upregulation of hnRNPC1/C2 expression, which may induce vitamin D deficiency by inducing VDR and trophoblast dysfunction 117 . Furthermore, METTL3 was found to facilitate the maturation of miR-497-5p/195-5p by mediating DGCR8's recognition of pri-miR-497-5p/195-5p in an m 6 A-dependent manner. Increased miR-497-5p/195-5p in the preeclampsia-affected placenta was found to suppress WWP1 expression, disturbing trophoblast proliferation, migration, and invasion, eventually aggravating preeclampsia progression 118 . The pathogenesis of recurrent miscarriage (RM) has not been fully elucidated yet. In addition to the known factors including genetic, endocrine, anatomical, and immunological disorders 119 , epigenetic abnormalities are also part of the underlying mechanisms 120 . So far, the research on the role of m 6 A in RM has mainly focused on its effects on trophoblast function. Higher ALKBH5 expression and lower m 6 A levels were found in chorionic villi from RM patients. ALKBH5 diminished m 6 A modification of CYR61 mRNA, reducing its stability and subsequent protein expression. These changes inhibited the proliferation and invasion of trophoblast cell at the maternal-fetal interface in early pregnancy, suggesting that ALKBH5 may be a potential key factor in the pathogenesis of RM 121 . The m 6 A modification was also found to be involved in BPDE-related RM. BPDE, a metabolite of environmental benzo(a)pyrene, was found to upregulate lncHZ01 in trophoblast cells, and lncHZ01-meditated MXD1 upregulation promoted METTL14 transcription, which increased m 6 A modification and enhanced the stability of lncHZ01, forming a positive feedback loop that eventually inhibited trophoblast cell proliferation and induced miscarriage 122 . Higher m 6 A levels and several aberrantly expressed m 6 A regulators were found in spontaneous abortion (SA) patients. FTO, in particular, was significantly downregulated in the chorionic villi and trophoblasts of SA patients. Lower FTO changed the m 6 A patterns of several genes involved in immunotolerance, immune cell infiltration and angiogenesis, demonstrating a possible role of m 6 A in SA progression 123 .

More than 170 kinds of RNA modifications were found to be widely distributed among various organisms 1 , which are collectively known as the 'epitranscriptome'. Substantial studies aimed to uncover the mechanisms and characterizations of these modifications, such as N6-methyladenosine (m 6 A), N1-methyladenosine, 5-methylcytidine and pseudouridine 2 , unveiling a brand-new landscape of epigenetic regulatory mechanisms. Among all of these modifications, m 6 A, which has been reported as early as 1974 3 , 4 , was considered to be the most prevalent chemical modification in eukaryotic messenger RNAs 2 , 5 , 6 . The m 6 A modification is preferentially enriched around stop codons, 5'- and 3'-untranslated regions (UTR), as well as in long internal exons with the consensus motif RRACH (R = A or G, H = A, C or U) 7 , 8 . Due to its involvement in various RNA-related bioprocesses, including RNA decay, splicing, transport and translation 9 - 12 , m 6 A is a pivotal regulator of RNA fate and plays an irreplaceable role in cell differentiation, development, metabolism, stress response and other cellular activities 2 , 13 - 15 . Epigenetic molecules and signaling pathways in the female reproductive system have various effects and are important for female fertility 16 - 19 . With the deepening of m 6 A research, there is emerging evidence for the involvement of m 6 A in various physiological and pathological processes in the female reproductive system, including oogenesis and diseases, subsequently affecting female fertility. Here, we introduce recent advances in research on m 6 A regulators and their functions, and highlight the role of m 6 A in oogenesis and female reproductive diseases, as well as the underlying mechanisms driving these processes.

The introduction of m 6 A into transcripts mainly occurs in the nucleus, making it a nuclear imprint of RNA fate 55 , 56 . The m 6 A levels determined by the m 6 A MTC and m 6 A erasers re closely related to RNA metabolism outcomes in the cytoplasm. Through reversible and dynamic methylation of adenine residues, m 6 A regulates the stability, splicing, transport and translation of RNAs 21 , 22 , playing an important role in a variety of physiological and pathological processes. Possible involvement of m 6 A modification in regulating RNA stability was first explored in 1978. Researchers deduced that rapid elimination of m 6 A in the cytoplasm may be caused by the shorter half-life of mRNAs with a high m 6 A content 57 . This hypothesis that mRNAs harboring more m 6 A were susceptible to degradation has been proved later, and Mettl3 knockout, which significantly decreased the m 6 A levels of RNAs, remarkably increased their half-life 58 . There is substantial evidence that m 6 A readers are the main regulators of RNA stability. RNA decay takes place in cytoplasmic processing bodies (P bodies) 59 , and YTHDF2 was found to colocalizes with P bodies and its cognate mRNAs were intended to have shorter half-live, demonstrating YTHDF2's role in destabilizing its mRNA targets. The deadenylation-dependent decay pathway was also a part of mechanism driving YTHDF2-mediated RNA decay 12 , 42 . YTHDF3 shares targets with its family members and promotes RNA degradation in cooperation with YTHDF2 44 . Conversely, IGF2BPs enhances mRNA stability by recognizing m 6 A modifications, increasing the abundance of its mRNA targets. IGF2BPs silencing led to comprehensive downregulation of its target genes 54 , 60 . It is worth mentioning that m 6 A modifications are also deposited in the RNAs of pseudogenes. These RNAs have higher m 6 A levels than their cognate mRNAs, which facilitates the degradation of processed pseudogene transcripts in the cytoplasm. Given that processed pseudogenes cannot be recognized and cleaned by canonical nonsense-mediated decay process, m 6 A-meditated cytosolic elimination of these nonfunctional RNAs provides a novel mechanism of RNA surveillance 61 . The m 6 A modification plays a key role in sex determination in Drosophila by modulating the splicing of Sex lethal transcripts 62 - 64 , revealing a novel posttranscriptional regulatory mechanism of gene expression. However, the effects and functions of m 6 A modification in RNA splicing in mammals have not been fully elucidated. Some researchers proposed that m 6 A does not have a major effect on splicing 58 . However, there is accumulating evidence that m 6 A modification is involved in RNA splicing through multiple mechanisms. Mettl3 -depletion partially affected RNA splicing in terms of frequency and type of alternative splicing events 15 . The demethylases FTO and ALKBH5 control this process by regulating m 6 A levels in alternative exons cassettes and coding sequences, respectively 36 , 65 . The nucleus-located m 6 A reader, YTHDC1, participates in the processing of transcripts together with splicing regulators such as CPSF6, SRSF3, and SRSF7 10 , 66 . Mechanistically, the distribution pattern of m 6 A modifications may be a key factor driving the impact of m 6 A on alternative splicing. It was found that m 6 A is preferentially located in long exons, near the stop codons, and in 3′-UTR 48 . Yet another study found that m 6 A was enriched near splice sites in both exonic and intronic regions 67 . This differential distribution may lead to region-specific binding of splicing-related proteins, thereby mediating alternative splicing. The indirect effect of m 6 A on RNA splicing should also be taken into consideration. By controlling the expression of highly m 6 A-modified splicing regulators such as SON44, HNRNPC38 and HNRNPF 68 , m 6 A may also alter RNA splicing outcomes without direct participation. Correct subcellular localization of transcripts is closely related to their function, making m 6 A modification an important regulator of RNA fate by affecting nuclear RNA export. A conditional knockout of Mettl14 led to nuclear retention of m 6 A-modified mRNAs by impairing the preferential binding of FMRP 69 . ALKBH5 -deficient cells with increased m 6 A levels showed accelerated nuclear export and massive cytoplasmic accumulation of mRNA, leading to significant changes in the ratio of nuclear and cytoplasmic mRNA content 9 .The m 6 A reader YTHDC1 was found to enhance the binding of m 6 A-modified RNAs to nuclear export adaptor protein SRSF3 and mRNA export receptor NXF1, which facilitates the nuclear export of its target mRNAs 46 . These studies proved the involvement of m 6 A in nuclear RNA export, but its effect on the whole transcriptome needs to be further elucidated. Some researchers have shown that m 6 A facilitates mRNA translation by interacting with eukaryotic translation initiation factor eIF3 via two different pathways 11 , 70 . As one of the most important translation initiation factors in eukaryotic cells eIF3 is usually bound to the 5′ untranslated regions of mRNAs and recruits the small ribosomal subunit around its binding sites, thereby regulating translation initiation 71 . It has been shown that the effect of the m 6 A reader YTHDF1 on protein expression is dependent on the eIF3-related translation machinery. YTHDF1-binding mRNAs showed a higher ribosome-bound fraction and translation efficiency 11 . Conversely, YTHDF1 silencing led to significantly downregulated protein production of its target mRNAs. Since YTHDF1 usually binds RNA close to stop codons in mammalian cells, the effect of 3'UTR-enriched YTHDF1 was based on eIF4G-induced spatial proximity to translation initiation sites 11 . Direct eIF3 recruitment around 5'UTR also initiated mRNA translation. Cell-stress associated mRNAs contain m 6 A modifications around the 5'UTR region and exhibit enhanced cap-independent translation 70 . Additionally, YTHDF1 was found to augment the translation of EIF3C in an m 6 A-dependent manner and form a YTHDF1-EIF3C axis promoting general protein synthesis 41 . Furthermore, METTL3, which was generally known as a methylase acting in the nucleus, also took part in mRNA regulation by interacting with the translation initiation machinery. Cytoplasmic METTL3 physically and functionally associates with eIF3 subunits, bridging its binding sites to the 5′-end of the mRNA to meditate mRNA circularization 72 , 73 . METTL3 depletion remarkably downregulated the expression of its target mRNAs 73 . In addition to mRNAs, the deposition of m 6 A modifications also affects the fate of modified non-coding RNA (ncRNA) in various ncRNA-related bioprocesses 74 . METTL3 induced m 6 A hyper-methylation facilitated stabilizing lncRNA, thus enhancing its expression 75 , 76 . FTO decreased the m 6 A level on the LINC00022 transcript and inhibited its degradation via the m 6 A reader YTHDF2 77 .HNRNPA2B1, a nuclear m 6 A reader, showed a METTL3-like effect in RNA splicing and promoted primary miRNA processing by interacting with the microRNA microprocessor complex protein DGCR8 50 . YTHDC1 was involved in the circRNA transport process. YTHDC1 silencing led to significant nuclear retention of circNSUN2, which was rescued by enforced expression of wild-type YTHDC1 78 . Moreover, the m 6 A modifications can initiate circRNA translation through YTHDF3-mediated recruitment of the translation initiation complex 79 . The m 6 A modification plays a key role in modulating interaction between ncRNAs and their targeted RNAs. For example, m 6 A modification promoted miR-133a binding to its targets and enhanced the miR-133a repression 80 . LncRNA ILF3-AS1 recruited METTL3 to ILF3 mRNA, which increased its m 6 A level and strengthened its stability 81 . The m 6 A-dependent RNA structural changes regulate the accessibility of RNA binding motifs of proteins to affect RNA-protein interactions, which is termed as “m 6 A-switch”. The m 6 A modification destabilizes hairpin stem structure of lncRNA MALAT1, thus making it more accessible for the RNA binding proteins 48 .

The m 6 A modification is a reversable process dynamically controlled by m 6 A writers and erasers, and its variable downstream effects rely on the recognition of m 6 A modifications by m 6 A readers (Figure 1 ) . The introduction of the m 6 A modification is accomplished by the multicomponent methyltransferase complex (MTC). The MTC consists of the catalytic subunit METLL3 and auxiliary subunits including METTL14, WTAP, KIAA1429, RBM15 and ZC3H13 20 . METTL3 serves as the core component of the MTC by installing the m 6 A modification by adding a methyl group to the N6 position of adenine. METTL14 forms a stable heterodimer core complex with METTL3, enabling the latter to recognize its substrates 21 - 23 . WTAP is indispensable for the correct localization of the METTL3-METTL14 complex in the nuclear speckles, which guarantees the correct continuation of m 6 A-related processes 24 , 25 . KIAA1429 and RBM15 maintain the m 6 A levels by recruiting the MTC to specific RNA regions 24 , 26 , 27 . ZC3H13 and its Drosophila homolog Flacc function as a bridge between the MTC and its mRNA targets, and guide RMB15 to WTAP to facilitate m 6 A modification 28 , 29 . Hakai is a newly identified member of the MTC in Drosophila and human cells that helps stabilize other subunits of the MTC through its ubiquitination domain 30 . Given that METTL3 only produces a part of RNA m 6 A modifications, there must be other writers meditating the other methyl transfer reactions 31 . METTL16 is a newly identified m 6 A methyltransferase targeting pre-mRNAs and various non-coding RNAs 32 , 33 . It consists of two structural domains, whereby the N-terminal methyltransferase domain independently recognizes the 5′-UACAGAGAA motif and the C-terminal vertebrate conserved region binds to internal stem-loops that facilitate the methyltransferase activity of METTL16 34 . In addition, METTL5 and ZCCHC4 were found to be m 6 A writers that respectively act on the 18S and 28S rRNAs in an MTC-independent manner. METTL5 forms a heterodimer with TRMT112 to maintain its metabolic stability and serves as an exclusive m 6 A rRNA methyltransferase. 35 Erasers are demethylases that remove m 6 A modifications in a ferrous iron and α-ketoglutaric acid dependent manner, dynamically regulating the m 6 A labeling of mRNAs and other types of transcripts in the nucleus. Erasers determine the m 6 A levels of transcripts together with m 6 A writers, thus modulating the subsequent effects of m 6 A readers. There is evidence that FTO controls RNA splicing by modulating he interaction of RNAs with SRSF2 protein 36 . ALKBH5, another known m 6 A eraser, has been proved to colocalize with mRNA-processing factors in nuclear speckles, and it plays critical roles in RNA transport, synthesis and stability. ALKBH5-dependent demethylation precisely controls the splicing process and length of the 3'-UTR region 37 . Recognition and binding of m 6 A readers to their target RNAs is required for m 6 A modification to execute its important functions in regulating RNA fate. The m 6 A readers discovered to date include YT521-B homology (YTH) domain-containing proteins, heterogeneous nuclear ribonucleoproteins (HNRNPs), insulin-like growth factor 2 mRNA-binding proteins (IGF2BPs) and eukaryotic initiation factor 3 (eIF3) 38 , 39 . These m 6 A readers take part in different RNA metabolic processes and affect different aspects of RNA behavior. YTH domain-containing proteins, the most researched m 6 A readers, recognize m 6 A modifications through the YTH domain and subsequently participate in various RNA-related processes 40 . YTHDF1 positively regulates the translation of its targeted RNAs in a m 6 A-denpend manner 11 , 41 , YTHDF2 selectively induces the degradation of m 6 A-modifed mRNAs, decreasing the abundance of its targets 12 , 42 , while YTHDF3 facilitated decay and translation of RNAs by interacting with YTHDF1 and YTHDF2 43 , 44 . As a nuclear m 6 A reader, YTHDC1 meditates nuclear RNA export and is required for alternative splicing 45 , 46 . YTHDC2 promotes translational activities and also accelerates the degradation of its targets, showing a double-sided effect 47 . HNRNPs and IGF2BPs are also protein families that function as m 6 A readers. Specifically, the binding affinity of HNRNPC for transcripts can be increased by m 6 A-mediated RNA structure alteration, leading to the m 6 A-switch effect 48 . HNRNPA2B1 binds RNA through its RNA recognition motif and recognizes m 6 A-modified RNA in a non-specific manner, enhancing METTL3-dependent microRNA processing and production 49 , 50 . HNRNPG interacts with the phosphorylated carboxy-terminal domain of RNA polymerase II through its RGG motifs and co-transcriptionally regulates alternative splicing 51 . IGF2BPs are a group of RNA binding proteins that selectively bind m 6 A-modified RNA and share overlapping RNA targets. IGF2BPs enhance mRNA stability and promote mRNA translation, thus upregulating gene expression 52 - 54 .

The research on m 6 A RNA modification has expanded broad horizons for researchers and uncovered several new epigenetic mechanisms underlying a number of pathophysiological processes. This reversible modification is controlled by three kinds of regulators and influences various RNA-related processes such as RNA degradation, splicing, transport and translation, regulating gene expression from multiple aspects at the post-transcriptional level. The involvement of m 6 A modifications has been observed in various bioprocesses including cell differentiation, development, metabolism, stress response and other cellular activities 2 , 13 - 15 , serving as an important epigenetic regulatory mechanism underlying both physiological and pathological processes. In the female reproductive system, m 6 A modifications regulate multiple stages of oogenesis, including follicle development, oocyte maturation, and the maternal-to-zygotic transition, as well as the self-renewal of female germline stem cells artificially established in vitro . Furthermore, m 6 A participates in the pathogenesis of a variety of female reproductive diseases including endometriosis and adenomyosis, polycystic ovary syndrome, premature ovarian insufficiency, preeclampsia, and recurrent miscarriage, all of which underscore the impact of m 6 A on female fertility. Recently several researches have revealed the clinical potential of m 6 A-targeting strategies. METTL3 inhibitors exhibited the promising anti-cancer effect against acute myeloid leukemia 124 , 125 . pharmacological inhibition of FTO significantly suppressed leukemia stem/initiating cell self-renewal and sensitized leukemia cells to T cell cytotoxicity 126 . However, m 6 A was only considered as a prospective therapeutic target of cancer treatment in current evidences 127 . Considering its crucial participation in female gonad development and reproductive diseases, m 6 A-targeting interventions should also be taken into account as promising approaches in treatment of infertility and reproductive disorders. Nevertheless, our understanding of the full impact of m 6 A is far from complete. For example, how different m 6 A readers recognize m 6 A-modified transcripts and lead to different transcript outcomes is still unclear. More importantly, translational studies aiming to utilize m 6 A as a novel target for therapy are in their infancy. All these unsolved questions merit further studies in the future.