Abstract
In brief
RNA modifications play key roles in regulating various biological processes. This article discusses and summarizes the recent advances of RNA m6A modifications related to mammalian gametogenesis, early embryonic development, and miscarriage.
Abstract
The epitranscriptome is defined as the collection of post-transcriptional chemical modifications of RNA in a cell. RNA methylation refers to the chemical post-transcriptional modification of RNA by selectively adding methyl groups under the catalysis of a methyltransferase. The N6 methyladenosine (m6A) is one of the most common of the more than 100 known RNA modifications. Recent research has revealed that RNA m6A modifications are reversible. Additionally, m6A containing RNA can be selectively identified by immunoprecipitation followed by high-throughput sequencing (MeRIP-SEQ). These two developments have inspired a tremendous effort to unravel the biological role of m6A. The role of RNA m6A modifications in immune regulation, cell division, stem cell renewal, gametogenesis, embryonic development, and placental function has gradually emerged, which is of great significance for the study of post-transcriptional regulation of gene expression in reproductive biology. This review summarizes the current knowledge about RNA m6A modification in a variety of mammalian reproductive events.
Introduction of RNA m6A modifications
Epigenetics refers to the study of heritable changes in gene expression that are not based on altered DNA sequences, such as DNA methylation, histone modification, maternal effects, genome imprinting, and RNA editing. RNA modifications can contribute significantly to this phenomenon, and a collective name for all RNA modifications is the ‘epitranscriptome’. More than 100 RNA modifications have been identified in eukaryotic RNA, of which N6 methyladenosine (m6A) is the most abundant type and is both dynamic and reversible (Machnicka et al. 2013, Zhao et al. 2017). The reversible m6A modification in mRNA is mostly identified in the RRACH consensus sequence (R=A/G; H=A/C/U) and is essential for fertility, growth, and development of mammals (Wei & Moss 1977, Harper et al. 1990, Meng et al. 2019, Hu et al. 2020).
Three types of proteins have been identified that affect RNA m6A modifications: methyltransferases, demethylases, and m6A-binding proteins, known as ‘writer’, ‘eraser’, and ‘reader’ proteins, respectively. The m6A methyltransferase complex is composed of methyltransferase-like 3 (METTL3), methyltransferase-like 14 (METTL14), Wilms’ tumour 1-associating protein (WTAP), and KIAA1429 (also known as vir-like m6A methyltransferase-associated protein). The m6A methyltransferase plays a catalytic role to form RNA m6A modifications (Bokar et al. 1997, Liu et al. 2014, Ping et al. 2014, Schwartz et al. 2014). The discovery of the two m6A demethylases, fat mass and obesity associated (FTO) and alkB homolog 5 (ALKBH5), revealed the dynamic potential of m6As in mRNA and sparked interest in epitranscriptomic research (Jia et al. 2011, Zheng et al. 2013). In addition to adding and removing methyl groups through methyltransferase and demethylase, m6A must be recognized by a binding protein to convey its biological activity. These m6A-binding proteins, also known as ‘reader’ proteins, include YTH domain containing 1 (YTHDC1), YTHDC2, YTH m6A RNA-binding protein 1 (YTHDF1), YTHDF2, and YTHDF3, which bind to m6A through their carboxy-terminal YTH domain (Li et al. 2014, 2017, Xu et al. 2014, Zhu et al. 2014, Wang et al. 2015, Hsu et al. 2017). Dynamic m6A modifications are recognized by selected binding proteins, thus influencing the translation status and lifetime of mRNA. For example, YTHDF2 converts the m6A modification into an RNA turnover signal and transports bound RNA to the decay machinery (Sheth & Parker 2003, Wang & He 2014, Wang et al. 2014a ). Another binding protein, heterogeneous nuclear ribonucleoprotein C (HNRNPC), is in the nucleus and is responsible for recognizing the m6A-modified mRNA and mediating selective splicing of mRNA, while the m6A-binding protein HNRNPA2B1 has been shown to promote the conversion of pri-miRNA to pre-miRNA (Alarcón et al. 2015).
RNA m6A modifications have been shown to play roles in a variety of biological processes, including X-inactive specific transcript-mediated transcriptional inhibition (Patil et al. 2016), mRNA stability (Wang et al. 2014a ), mRNA splicing (Xiao et al. 2016), and translation efficiency (Wang et al. 2015). Moreover, studies have found that dynamic mRNA m6A modifications and epigenetic regulation mediated by m6A are associated with reproductive diseases such as infertility, spontaneous miscarriage, abnormal fetal growth, and development (Hsu et al. 2017, Meng et al. 2019, Sui et al. 2020, Qiu et al. 2021, Zheng et al. 2022) (Fig. 1). Here, we review and summarize the biological functions of m6A in mammalian gametogenesis and embryonic development. In addition, we also discuss the abnormal RNA m6A modifications in the pathogenesis of miscarriage and speculate about the possible roles of m6A in the regulation of parturition in mammals.
Working model of the cellular pathways of m6A modifications in nuclear RNA.
Citation: Reproduction 165, 1; 10.1530/REP-22-0112
Working model of the cellular pathways of m6A modifications in nuclear RNA.
Citation: Reproduction 165, 1; 10.1530/REP-22-0112
Working model of the cellular pathways of m6A modifications in nuclear RNA.
Citation: Reproduction 165, 1; 10.1530/REP-22-0112
The function of RNA m6A modifications in mammalian gametogenesis
METTL3 and METTL14 act synergistically in mammalian spermiogenesis
Methylation of adenosine to m6A is mediated by a complex composed of multiple proteins. METTL3, which is the active transferase in the complex, can catalyse the transfer of a methyl group from the cofactor S-adenosyl-L-methionine to the N6 position of adenosine alone (Liu et al. 2014). Importantly, METTL3 is highly conserved in eukaryotes, with homologous genes in yeast, plants, and fruit flies (Clancy et al. 2002, Zhong et al. 2008, Haussmann et al. 2016). METTL14 also belongs to the active transferases in the complex (Liu et al. 2014). Specifically, knockout of the Mettl3 gene in mouse germ cells with Vasa-Cre, which is expressed in spermatogonia, led to severe inhibition of spermatogonial differentiation and blocked the initiation of meiosis, by regulating the alternative splicing of genes related to spermatogenesis (Xu et al. 2017). Another research study published in the same year investigated mice in which Mettl3 or Mettl14 was deleted in advanced germ cells using Stra8-GFPCre. These advanced germ cells developed into spermatocytes that showed normal spermatogenesis (Lin et al. 2017). Notably, the deletion of both Mettl3 and Mettl14 with Stra8-GFPCre led to impaired spermiogenesis, due to the abnormal translation of transcripts that are required for spermatogonial stem cell proliferation and differentiation (Lin et al. 2017). The above-mentioned studies suggested that METTL3 may play different roles in spermatogonia and spermatocytes, while METTL3 and METTL14 act synergistically for spermatid differentiation in the late stages of spermiogenesis. The roles of methyltransferases in spermiogenesis are further supported by clinical research, which revealed that increased m6A content in sperm RNA attributed by METTL3 is a risk factor for asthenozoospermia (Yang et al. 2016), suggesting that either hyper- or hypo-m6A methylation will affect the spermatogonial process and subsequently male fertility. Although current research has mostly focused on the effects of the key m6A methyltransferase METTL3 in male spermatogenesis, our previous study revealed that deletion of Mettl3 in female mouse germ cells severely inhibits oocyte maturation by reducing maternal mRNA translation efficiency (Sui et al. 2020). Further, another study showed that oocyte-specific inactivation of Mettl3 with Gdf9-Cre induction affected RNA stabilization during oocyte growth, causing DNA damage accumulation in oocytes, defective follicle development, and abnormal ovulation (Mu et al. 2021), thus further adding evidence about the important role of METTL3 in male and female gametogenesis.
In mammals, studies have shown that WTAP interacts with METTL3 and METTL14 in the methyltransferase complex. Although WTAP exhibits no methyltransferase enzymatic activity, knocking down Wtap causes degradation of METTL3 and METTL14 and decreases the level of RNA m6A modification (Liu et al. 2014, Ping et al. 2014). While no studies have been conducted yet on the functions of WTAP in mammalian spermatogenesis, it was reported that WTAP plays a key role in meiotic maturation and developmental potency of pig oocytes, along with a higher incidence of spindle defects and chromosome misalignment if knocked down (Wang et al. 2018). Moreover, conditional knock out of Kiaa1429 in mouse oocytes reduces the level of m6A in oocytes and affects the alternative splicing of genes related to oogenesis. This results in female infertility with defective follicular development, which is caused by the inhibition of the breakdown of fully grown germinal vesicles in oocytes, and thus the ability to resume meiosis is lost (Hu et al. 2020).
ALKBH5 but not FTO plays a role in mammalian spermatogenesis
Both FTO and ALKBH5 belong to the non-haeme Fe(II)- and α-ketoglutarate (α-KG)-dependent dioxygenase AlkB protein family and are widely expressed proteins with m6A demethylase activity (Jia et al. 2011, Zheng et al. 2013). However, the biological effects of FTO and ALKBH5 are very discriminatory, which may be due to their tissue-specific expression or, more likely, different preferences for substrates. In mice, FTO is predominantly expressed in muscles and the brain, while ALKBH5 is highly expressed in the testes and lungs (Gerken et al. 2007, Zheng et al. 2013). Due to the differential expression of FTO and ALKBH5, the catalysed mRNA substrates may vary in different tissues and cells.
FTO is essential for the normal development of the human central nervous system and cardiovascular system (Gao et al. 2010, Äijälä et al. 2015, Mongelli et al. 2020). Nevertheless, loss of Fto in mice resulted in postnatal growth retardation and low body mass, whereas the reproductive system develops normally in mice lacking FTO (Fischer et al. 2009). Unlike FTO, ALKBH5 catalyses the reaction to remove methyl groups from m6A-modified adenosine, instead of oxidative demethylation (Zheng et al. 2013). In mice, ALKBH5 co-localizes with nuclear speckles and affects the assembly and modification of mRNA transcription factors. ALKBH5 is indispensable for mRNA methylation status and mRNA metabolism. Knocking out Alkbh5 in mice impairs spermatogenesis due to spermatocyte apoptosis during meiosis metaphase and aberrant mRNA splicing (Tang et al. 2018).
YTHDC1, YTHDC2, and YTHDF2 are essential for both male and female gametogenesis
In mammals, YTH domain-binding proteins have five ‘members’: YTHDF1, YTHDF2, YTHDF3, YTHDC1, and YTHDC2. Through the application of m6A sequencing, RNA affinity chromatography, and mass spectrometry, the binding sites of m6A readers were mapped (Dominissini et al. 2012, Wang et al. 2014a ). Although the structures of YTH proteins are very analogous to each other, the phenotypes of mice deficient in each protein are quite dissimilar, indicating that each YTH protein has a specific function and/or expression pattern (Wang & He 2014, Wang et al. 2015).
Depletion of Ythdc2 in mouse germ cells leads to infertility: females have smaller ovaries and males have smaller testes than their littermates, and further analysis shows that conditional knockout of Ythdc2 in mouse germ cells decreases mRNA translation efficiency and target mRNA abundance to reduce spermatogenesis (Hsu et al. 2017). Similarly, Ythdc1 depletion results in compromised fertility, impaired spermatogonia, and a reduced survival rate of male mice (Kasowitz et al. 2018). In female mice, oocytes lacking Ythdc1 show larger cytoplasmic RNA granules, splicing defects, and obvious increases in polyadenylation, which lead to oocyte maturation disorders and female infertility (Kasowitz et al. 2018). Conditional knockout of Ythdf2 in mouse germ cells disrupts the transcriptome of the round and elongated sperm cells and causes abnormal sperm morphology, impaired sperm motility, and loss of fertilization ability, which eventually leads to male sterility (Qi et al. 2022). In female mice, YTHDF2 is required for oocyte maturation via post-transcriptional regulation, which is essential for the activation of zygotic genomes, and female mice lacking Ythdf2 are sterile as well (Ivanova et al. 2017).
Through affinity chromatography, several additional m6A-binding proteins have been identified but need to be further clarified (König et al. 2010, Alarcón et al. 2015). The existence of m6A-binding proteins may be related to the basic function of m6A in various life activities especially in reproductive biology (Table 1).
Proteins related to RNA m6A methylation in reproductive biology.
| Function/protein name | Species | Physiological process | References |
|---|---|---|---|
| Methylation | |||
| METTL3 | Mouse | Spermatogenesis | Xu et al. (2017) |
| METTL3 | Mouse | Oocyte development | Sui et al. (2020), Mu et al. (2021) |
| METTL3 | Mouse | Embryonic development | Geula et al. (2015) |
| METTL3/METTL14 | Mouse | Spermatogenesis | Lin et al. (2017) |
| METTL14 | Mouse | Embryonic development | Meng et al. (2019) |
| WTAP | Pig | Oocyte development | Wang et al. (2018) |
| KIAA1429 | Mouse | Oocyte development | Hu et al. (2020) |
| Demethylation | |||
| FTO | Human | Spontaneous miscarriage | Qiu et al. (2021) |
| ALKBH5 | Mouse | Spermatogenesis | Tang et al. (2018) |
| ALKBH5 | Mouse | Spontaneous miscarriage | Zheng et al. (2022) |
| Methyl binding | |||
| YTHDC1 | Mouse | Oocyte development | Kasowitz et al. (2018) |
| YTHDC1 | Mouse | Embryonic stem cells | Chen et al. (2021) |
| YTHDC2 | Mouse | Spermatogenesis | Hsu et al. (2017) |
| YTHDF2 | Mouse | Spermatogenesis | Qi et al. (2022) |
| YTHDF2 | Mouse | Oocyte development | Ivanova et al. (2017) |
| YTHDF2 | Goat | Embryonic development | Deng et al. (2020) |
| YTHDF2 | Mouse | Embryonic development | Li et al. (2018) |
RNA m6A modification and mammalian embryonic development
In addition to the critical roles of RNA m6A modification in mammalian gametogenesis, a growing number of studies have shown that RNA m6A modification modulates mammalian embryonic development. m6A, both at the 5′-UTR and in the vicinity of the stop codon in placental mRNA, plays key roles in fetal growth (Taniguchi et al. 2020). Additionally, with the method of ultralow-input m6A RNA immunoprecipitation followed by sequencing, the RNA m6A modification was proven to have important functions in specific contexts during the maternal-to-zygotic transition (MZT), namely regulating maternal RNA stability in oocytes and timely RNA degradation during MZT (Wu et al. 2022).
METTL3 and its homologues affect mammalian embryonic development
Knocking out Mettl3 in mouse embryonic stem cells impedes its self-renewal ability and differentiation ability, suggesting that RNA m6A modification plays an indispensable role in the transformation process of mammalian embryonic stem cells (Batista et al. 2014, Wang et al. 2014b ). Mettl3 deletion in mice causes early embryonic lethality due to a failure to downregulate Nanog mRNA (Geula et al. 2015). Our recent study revealed that the deletion of Mettl3 in female mouse germ cells not only inhibits oocyte maturation but also impedes the subsequent MZT and zygotic genome activation, probably due to its interference in disrupting maternal mRNA degradation (Sui et al. 2020). Further, METTL3 played an essential role in the establishment of m6A on zygotic genome activation transcripts, which subsequently promoted the timely turnover of two-cell specific markers and ensured the developmental progression of pre-implantation embryos (Wu et al. 2022). Deletion of the METTL3 gene results in embryonic arrest during the morula to blastocyst transition and developmental defects in porcine trophectoderm cells. Furthermore, METTL3-mediated m6A methylation has been shown to negatively regulate autophagy in support of blastocyst development (Cao et al. 2021). In addition, conditional Mettl14 knockout mice, constructed with the CRISPR-Cas9 system, developed embryonic retardation and morphological abnormalities at 6.5 days post-coitus, in which the development and maturation of the ectoderm are blocked, mainly due to differentiation resistance (Meng et al. 2019). In zebrafish, the complex formed by METTL3, METTL14, and WTAP plays roles in regulating mRNA transcription and alternative splicing, which is indispensable for tissue differentiation as well (Ping et al. 2014). These studies suggest that methyltransferases are evolutionarily highly conserved and are particularly important for embryonic development.
ALKBH5 and FTO in embryonic development
ALKBH5 catalyses m6A demethylation in mRNA and plays a key role in the regulation of physiological functions such as testicular development in mice (Zheng et al. 2013). As ALKBH5 is highly conserved in eukaryotes, it is reasonable to speculate that ALKBH5 has a similar function in humans, regulating testicular development in men (Landfors et al. 2016). ALKBH5 also controls trophoblast invasion at the maternal–fetal interface by regulating the stability of cysteine-rich angiogenic inducer 61 (CYR61) mRNA (Li et al. 2019), which may subsequently affect the embryonic development in utero. A recent study demonstrated that FTO regulates chromatin-associated long-interspersed element-1 RNA expression through RNA m6A demethylation and chromatin modification levels affecting the chromatin state at nearby sites. These modifications alter the proliferation and differentiation of mouse embryonic stem cells and embryonic development (Wei et al. 2022). Additionally, the homozygous mutant FTO protein (Arg316Gln) causes abnormalities such as rare growth retardation and developmental delay syndrome (Boissel et al. 2009, Daoud et al. 2016), suggesting that FTO also plays an important role in embryonic development.
YTHDF2 and YTHDC1 modulate RNA metabolism and affect embryonic development
YTHDF2 is essential for early embryogenesis as it advances maternal mRNA clearance, which might be through the recruitment of deadenylases and mRNA decapping enzymes. In YTHDF2 gene knockout goat embryos at the eight-cell stage, the maternally encoded mRNA decay and zygotic genome activation that depend on maternal mRNA clearance were impaired due to the specifically decreased expression of deadenylases and decapping enzymes (Deng et al. 2020). Depletion of Ythdf2 in mice causes lethality at late embryonic developmental stages, with embryos characterized by compromised neural development (Li et al. 2018). YTHDC1 has been shown to play essential roles in the self-renewal and differentiation potency of mice embryonic stem cells (Chen et al. 2021). Moreover, YTHDC1 is also essential for the processing of pre-mRNA transcripts in the oocyte nucleus and may play a similar non-redundant role throughout fetal development. Thus, YTHDC1 plays an essential role in embryo viability and germline development in mice (Kasowitz et al. 2018).
Cycloleucine is a methylation inhibitor, and cycloleucine treatment effectively reduced m6A levels, significantly reduced the ratio of four-cell embryos to blastocysts, disrupted normal lineage allocation, and increased apoptosis and autophagy levels in pig blastocysts (Yu et al. 2021). Further, RNA m6A methylation appeared to be critical for early embryonic development, and abnormal nuclear transfer-mediated m6A methylation reprogramming is involved in the regulation of early embryonic development in pigs.
RNA m6A modification and mammalian miscarriage
Spontaneous miscarriage is the most common complication in early pregnancy, with an incidence of 10–15% (Romero et al. 2014, Quenby et al. 2021). The maternal–fetal interface is the site of circulation and nutrient exchange between the placenta and growing foetus and is related to miscarriage and preeclampsia (Jauniaux et al. 2003). The Alkbh5-specific knockdown in the mouse placenta inhibited the invasion of trophoblast cells and led to a significant rate of foetus abortions in vivo, and conversely ALKBH5 has been shown to promote the invasion of trophoblasts (Zheng et al. 2022). In humans, downregulation of FTO causes markedly higher RNA m6A methylation and oxidative stress in the chorionic villi and disrupts immune tolerance and angiogenesis at the maternal–fetal interface, which ultimately leads to spontaneous miscarriage (Qiu et al. 2021). Obesity-related FTO rs9939609 single nucleotide polymorphism (SNP) is associated with recurrent miscarriage (Andraweera et al. 2015, Hubacek et al. 2016). Therefore, in clinical examination and treatment, the SNP of FTO may be useful in predicting the risk of recurrent miscarriage.
As we have mentioned before, the reduction of ALKBH5 in the trophoblastic layer increases the half-life of CYR61 mRNA and promotes steady-state CYR61 mRNA expression levels (Li et al. 2019), which provides a clue for further exploration of a wide range of RNA epigenetic regulation patterns not only in embryonic development disorders but also when recurrent miscarriages have occurred.
Perspectives
Reproduction is one of the key features of life and one of the most tightly regulated biological processes in the mammalian life cycle. It begins with gametogenesis, which involves the process of meiosis to form functional gametes from parent diploid cells, followed by the fertilization of oocytes and sperm, and subsequent development of the embryo and placenta which ends with parturition. At specific stages of mammalian gametogenesis and fertilized oocyte development, transcription and translation are stopped, and post-transcriptional modifications such as RNA m6A methylation play important roles to ensure correct gene translation and timely progress of reproductive processes. Dynamic RNA m6A modification is orchestrated by its methyltransferases, demethylases, and m6A-binding proteins. Due to ethical considerations, research on RNA m6A modifications cannot be fully applied to humans. By studying mammalian animal models such as mice, along with other organisms like zebrafish and fruit flies, RNA m6A modifications were found to play vital roles in gametogenesis, embryonic development, and placental function. Due to the high conservation of RNA m6A modification-related enzymes in mammals, we preliminarily speculated that RNA m6A modification may also be involved in similar processes in human, thereby affecting human reproductive health. However, further scientific experiments are needed to confirm these hypotheses.
Recently, we and other groups have verified that fetal lungs produce signals to initiate labour as they mature (Gao et al. 2015, Chen et al. 2020), and abundant RNA m6A modifications exist in the lungs of mammals (Xiao et al. 2019, Xiong et al. 2021). Further, RNA m6A modification regulates the lung fibroblast-to-myofibroblast transition via modulation of KCNH6 (potassium voltage-gated channel, subfamily H member 6) mRNA translation (Zhang et al. 2021). Therefore, it is reasonable to speculate that RNA m6A methylation may regulate fetal lung development, thus affecting normal delivery or causing premature labour, which is a topic worthy of attention and further research.
In view of the biological functions of RNA m6A modification on mammalian reproduction, using m6A as a target for the early screening, diagnosis, and treatment of infertility and abnormal parturition appears to be feasible. For example, the expression level of the ALKBH5 gene in men with abnormal semen may help to explore the cause of infertility, and manipulation of this gene may provide a therapeutic target for treatment. Precise treatment of m6A-related gene targets, including but not limited to METTL3, METTL14, WTAP, KIAA1429, FTO, YTHDC1, YTHDC2, and YTHDF2, by regulating RNA m6A modifications, can potentially ensure normal gametogenesis, which has a therapeutic effect on infertility. Since miR-186, miR-4429, miR-600, and STM2457 have been shown to target METTL3 mRNA and inhibit its expression (Zeng et al. 2020, Yankova et al. 2021, Wu et al. 2022), these three micro-RNAs and STM2457 can be used in the treatment of gametogenesis disorders (Liu et al. 2021). FTO inhibitors are used in patients with FTO gene abnormalities. Rhein is a competitive inhibitor of the FTO substrate, which blocks the binding of the single-stranded RNA substrate and FTO active centre and can inhibit the demethylase activity of mRNA m6A by FTO, so as to carry out treatment (Huang et al. 2021, Zannella et al. 2021). Further, FTO rs9939609 SNP may be a pathogenic factor for recurrent miscarriage (Andraweera et al. 2015), and this result requires further exploration, as it is involved in the strict control of important factors such as body mass index. Collectively, RNA m6A modifications may play key roles in pregnancy and are likely to be new potential therapeutic targets for infertility and abnormal parturition, although more strictly designed randomized control trial studies are obviously needed before m6A can be used in clinical therapies.
In contrast to DNA methylation and histone modification, RNA m6A epigenetic studies are just beginning and increasing in frequency. Such studies provide a new perspective for the prediction and treatment of reproductive diseases such as infertility and abnormal parturition, but the molecular mechanisms and specific targets still need future investigation. For instance, to understand the details of RNA m6A regulation, it is necessary to characterize RNA m6A levels in various species, cell types, and stages of development. Hopefully, future development of direct sequencing of RNA m6A will provide convincing evidence to understand its dynamics and functions in vivo. Additionally, it has been suggested that high sensitivity mass spectrometry will be the most accurate method to quantify m6A modifications. These perspectives in technology will further help us to understand the roles and mechanisms of m6A modifications in human health and disease.
Declaration of interest
The authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of this review.
Funding
This work was supported by National Natural Science Foundation of China No. 82120108011 (L G); Major Project of Shanghai Municipal Education Commission’s Scientific Research and Innovation Plan No. 2021-01-07-00-07-E00144 (L G); and Strategic Collaborative Research Program of the Ferring Institute of Reproductive Medicine, Grant No. FIRMA200502 (L G).
Consent for publication
All authors have read through the manuscript and approved the publication.
Author contribution statement
L G and A K conceived the project. X S wrote the paper. L G and A K revised and edited in English.
Acknowledgement
We would like to thank Editage (www.editage.com) for English language editing.
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