From the perspective of epigenetics, the regulatory mechanism of synonymous mutations on MAOA activity and structure was explored.

Monoamine Oxidase A (MAOA) is a metabolic enzyme responsible for the breakdown of neurotransmitters such as dopamine, norepinephrine, serotonin, and other monoamines. These monoamines are considered pivotal neurotransmitters in the pathophysiology of conditions like schizophrenia (Sun et al., 2012). MAOA has been consistently associated with conditions such as addiction, depression, autism, and antisocial behavior (Byrd and Manuck, 2014; Kolla and Bortolato, 2020). Additionally, MAOA plays a critical role in regulating neurotransmitters in the brain that are closely linked to emotions, including depression, reward, and aggression (Cases et al., 1995). Since the mid-1990s, researchers have been investigating the associations between genetic variants and various phenotypic abnormalities (Manuck et al., 2000). Among these polymorphisms, one of the primary focuses has been on rs1137070, which has been found to be associated with susceptibility to smoking (Shen et al., 2019), heroin addiction (Sun et al., 2017), hostility, and depression (Yen et al., 2021), as well as susceptibility to Parkinson’s disease (Sun et al., 2014), and autism (Verma et al., 2014), etc.

The rs1137070 is a C to T substitution at position 1,460 of the DNA sequence (c.1460C > T) in Maoa exon 14. This synonymous single nucleotide polymorphism (SNP) results in a synonymous change (Asp470Asp) and is located in the coding region of the X chromosome. A 2019 study found that polymorphisms in Maoa (rs1137070) play a moderating role in the effects of smoking (Shen et al., 2019). Other studies on rs1137070 demonstrated that individuals carrying the Maoa EcoRV polymorphism C allele (rs1137070; those with the 1460C > T polymorphism) in exon 14 had lower MAOA activity (Agatsuma et al., 2006). However, these studies on Maoa gene polymorphisms have primarily relied on questionnaires, brain imaging, and physiological and biochemical analyses, with the exact underlying mechanism remaining unclear. In addition, there is a lack of research on rs1137070 in cells and animals, so it is necessary to construct relevant cell and animal models to further complement the molecular mechanism of rs1137070 locus affecting MAOA.

Synonymous mutation refers to a mutation in which the codon is different but does not change the encoded amino acid. Increasing evidence indicates that synonymous codons are not used randomly or equally (Hanson and Coller, 2018). Concerning genetic polymorphisms of synonymous codons, studies have identified a common phenomenon known as codon usage bias (CUB), which involves the differential usage of synonymous codons of varying frequencies and is prevalent across various species (Liu et al., 2021b). It is now recognized that codon usage affects gene expression by regulating factors such as elongation speed, translation efficiency, initiation, termination, and accuracy. There have been relatively few animal experiments aimed at investigating the contribution of synonymous mutations to expression regulation or comprehensively assessing their impact on organisms. Therefore, despite the findings from population studies linking synonymous codon mutations to a variety of mood disorders, this paper aims to further elucidate the differential regulatory mechanisms of transcription and translation caused by synonymous mutations by constructing base-editing model animals.

The CRISPR/Cas9 system has gained widespread recognition for its utility in targeted insertion of exogenous DNA sequences, offering an effective method for creating animal models with synonymous mutations (Banan, 2020). In recent years, various genome editing tools have been developed to facilitate the creation of site-specific DNA double-strand breaks (DSBs). Examples include zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and the clustered regularly spaced short palindromic repeats CRISPR-Cas9 systems (Gaj et al., 2013). Cas9 can bind to multiple single-guide RNAs (sgRNAs), enabling efficient multiplexed genome editing in mammalian cells. This technology has found extensive use in the development of animal models featuring synonymous mutations in genes (Albadri et al., 2017; Kim et al., 2020). Cas9-engineered mice have become valuable assets in biological and disease modeling (Platt et al., 2014).

Genome-wide association studies (GWAS) have revealed substantial differences in the frequency distribution of this gene among different racial populations (Sirugo et al., 2019). Therefore, the primary objective of this study is to unravel the impact of genetic diversity within the human Maoa gene on gene biosynthesis and function through the creation of synonymous mutations in mice Maoa at position 1,409 T > C. To achieve this, we employed the CRISPR-Cas system to introduce the 1,409 T > C mutation into the mouse Maoa gene, homozygous gene mice with KI were obtained. This research aims to investigate the dysfunctional and molecular regulatory mechanisms of single nucleotide polymorphisms (SNPs) by assessing MAOA protein expression. We also seek to uncover the underlying mechanisms through which this synonymous mutation influences mRNA and protein structure, as well as its impact on transcriptional translation regulation. In summary, our findings offer fresh insights into the role of synonymous mutations in MAOA in the regulation of gene expression, shedding light on a new perspective for understanding the mechanism of MAOA enzyme activity. This research holds significant implications for understanding the pathogenesis of this gene mutation in the population and contributes to enhancing our comprehension of genetic diversity within the Chinese population.

Key resources are presented in Table 1.

Six-week-old C57BL/6 J Maoa-KI mice (synonymous mutations in mouse MAOA at position 1,409 T > C) were acquired from Gempharmatech Co., Ltd. Maoa-WT mice were acquired from Beijing Vital River Laboratory Animal Technology Co., Ltd. Both C57 Mouse were obtained and used as both embryo donors and foster strains. All experimental procedures were conducted in compliance with the protocols approved by the Laboratory Animal Management and Ethics Committee of China National Tobacco Quality Supervision and Test Center. These procedures adhered to established experimental practices and standards, and All animal experiments are comply with the ARRIVE guidelines and should be carried out in accordance with the U.K. Animals (Scientific Procedures) Act, 1986 and associated guidelines, EU Directive 2010/63/EU for animal experiments, or the National Research Council’s Guide for the Care and Use of Laboratory Animals. Mice were housed and bred in a specific pathogen-free (SPF) animal facility, with five mice per cage to minimize the occurrence of aggression and injuries. They were allowed to acclimate for 1 week before the commencement of experiments, during which they had free access to both food and water. The light and dark cycles were maintained at 12 h each, with a constant temperature of 25°C and humidity ranging between 40 and 60% in the facility. Mice were randomly assigned to different experimental groups. Mice were subjected to cervical dislocation for NSC collection Sampling was conducted between 9:00 and 15:00 from August to January (Kasowitz et al., 2018; Gupta et al., 2019; Liu et al., 2021a).

Maoa plays a pivotal role in various metabolic processes involving monoamines and holds significant importance in the metabolism of neuroactive and vasoactive amines across both the central nervous system and peripheral tissues. Moreover, Maoa serves as a crucial candidate for genetic investigations into various mental disorders (Ziegler and Domschke, 2018). The primary focus of this study is to delve into the mechanisms underlying the influence of Maoa gene polymorphisms and Maoa expression in individuals with T and C genotypes who are addicted to smoking, thereby shedding light on the role in addiction.

Based on GWAS and China Map data, it has been observed that different human ancestry exhibit two genotypes, C and T, for this gene, with varying frequencies (Cao et al., 2020). These genetic differences have been correlated with disparities in brain function (Shen et al., 2019). Further analysis of different populations found that the rs1137070 SNP was significantly associated with smoking susceptibility, heroin addiction, hostility and depression, Parkinson’s disease and autism, bipolar disorder, schizophrenia, and other disorders. Genome-wide analysis of the population also found that this locus was significantly associated with psychiatric disorders in Europeans (p = 0.00014, T allele OR = 1.466) and Asians (p = 0.015, OR = 1.148 for T alleles). By comparing amino acid sequences and protein structures, it has been discovered that Maoa in humans and mice share a high degree of similarity, with an impressive 87% homology (Figure 1B) and an exceptional 98% structural similarity (Figure 1C). Consequently, a mouse model featuring a synonymous mutant Maoa gene with a C base was developed using CRISPR-Cas9 technology, effectively simulating two gene polymorphisms observed in humans. In this research, the primary focus has been on investigating the Maoa gene from the perspective of gene expression regulation.

It’s noteworthy that SNPs are synonymous mutations, typically assumed not to influence gene expression. However, there is a growing body of evidence indicating that synonymous codons play a crucial role in determining gene expression levels and protein structure (Liu et al., 2021b). Synonymous mutations primarily impact gene expression by influencing transcriptional elongation speed, thereby regulating translation efficiency and accuracy. Hence, in the examination of Maoa gene expression, the study began by measuring the changes in MAOA levels and exploring whether the GAC codon in this sequence resulted in higher gene expression compared to the Maoa-WT group (Figures 2A–D). The findings demonstrated that both mRNA and protein levels of the Maoa gene were relatively lower in the CC genotype group compared to the WT group at 6 weeks of age. Subsequently, Maoa enzyme activity was assessed, revealing that the CC genotype displayed lower MAOA enzyme activity (Figure 2E), consistent with previous findings (Agatsuma et al., 2006). It’s worth noting that synonymous mutations can alter protein folding due to differences in codon translation speeds (Yu et al., 2015), potentially leading to changes in enzyme activity. Given that dopamine metabolism in rodents is primarily influenced by Maoa (Paterson et al., 1991), a decrease in MAOA protein levels and reduced enzyme activity can result in abnormal levels of dopamine in the brain. This mutation primarily affects Maoa epigenetically.

Further molecular biology experiments are needed to conclusively determine whether synonymous mutations affect MAOA expression through codon usage. Different organisms exhibit preferences for specific codons, with mammals often favoring C/G in the wobble position (Hershberg and Petrov, 2008). Therefore, we hypothesize that GAC is a more favorable codon compared to GAT. According to the codon usage bias theory, codon preference can influence gene expression, sometimes leading to increased protein production (Zhou et al., 2016) or, in other cases, decreasing it (Yang et al., 2019). Our Western blot (WB) and quantitative polymerase chain reaction (QPCR) results indicated that the GAC codon led to a downregulation of protein expression (Figures 2A–D). Therefore, in the best codon theory, we believe that GAT is the dominant codon compared to GAC, and GAT has a higher amount of gene expression products than GAC.

Translation dynamics are intricately linked with mRNA structure (Liu et al., 2021b) and protein structure (Zhou et al., 2015), and different gene sequences can impact mRNA secondary structure (Gu et al., 2013). Therefore, we initially explored whether GAC is a dominant codon, thereby affecting mRNA secondary structure and protein structural stability. We investigated the effects of synonymous codons on mRNA secondary structure, mRNA half-life, and protein half-life. The prediction of mRNA secondary structure using UNFold revealed that the KI group sequence had a higher number of pairable bases and formed smaller loops. Additionally, it had a lower Gibbs free energy compared to the four possible secondary structures of the WT sequence, theoretically rendering it more stable (Figures 3A–E). Some research has indicated that dominant codons are enriched in stable mRNA sequences, while rare codons are associated with the formation of mRNA with shorter half-lives. The prediction results of mRNA secondary structure align with this theory.

However, QPCR experiments revealed that while the Maoa-KI secondary structure was stable, its half-life was not longer. The half-life of the KI group (4.1667 h) was actually shorter than that of the WT group (4.556 h) (Figure 3F). This discrepancy in half-life suggests that mRNA structure stability may be constrained by other factors. Subsequent RNA-pull down experiments further illustrated that the regulation of RNA-binding protein (RBP) could influence mRNA structural stability (Figure 4E). Following this, WB experiments were conducted to measure protein half-life. The half-life of the WT group was 16.668 h, whereas the half-life of the KI group was 13.367 h, indicating that the protein stability of Maoa-KI was inferior (Figure 3G). This finding aligned with the results obtained from the MAOA protein amount ELISA detection (Figures 2C,D).

Dominant codons significantly affect translation efficiency, as they exhibit higher binding efficiency to ribosomes and tRNA, leading to faster translation. Translation speed also impacts the folding rate of translation, which, in turn, affects protein function and structural stability (Zhao et al., 2017). To explore whether GAC is indeed a dominant codon compared to GAT, a DNA sequence containing six GAC/GAT sites was selected to expand the binding difference. The results of the dual-luciferase experiment revealed that GAC produced a higher sea kidney luciferase fluorescence signal (Figure 4C), confirming that GAC is the dominant codon. While all codons influence translation rate, some codons can be rapidly decoded and exhibit high-efficiency binding to ribosomes, whereas others have lower ribosome binding efficiency due to lower tRNA concentration (Bae and Coller, 2022). The findings indicate that more stable mRNA secondary structures are more challenging to unwind, resulting in lower translation efficiency (Kaushik et al., 2018).

The prediction of Maoa’s secondary structure suggests that it has a more stable structure, making it harder to unfold during the translation process. Consequently, translation efficiency is lower, and because its mRNA and protein half-lives are shorter and more susceptible to degradation, overall protein expression in KI decreases (Figures 2C,D). These observations highlight significant differences in maintaining mRNA and protein stability among different synonymous codons. Furthermore, it is important to consider that optimal codons do not necessarily lead to upregulated protein and mRNA levels, as these can also be influenced by mRNA stability and post-translational modifications. However, it is important to acknowledge that these results have certain limitations at the animal level. It is possible that mRNA cleavage and post-translational modifications by RNA-binding proteins (RBPs) may have contributed to the decrease in expression observed in the study.

The study of molecular mechanisms in gene expression regulation encompasses various processes, including transcriptional and post-transcriptional regulation, translation initiation, and post-translational modifications that collectively determine mRNA levels, protein structure, activity, and stability (Chu et al., 2014). Transcriptional regulation is generally influenced by the strength of the promoter region and upstream regulatory elements, while protein translation is often regulated by post-translational modifications and protein degradation pathways. Synonymous mutations in the Maoa gene can indeed impact gene expression regulation. Previous research has highlighted several epigenetic regulatory mechanisms affecting MAOAexpression and activity. For instance, the Maoa promoter’s critical regions directly control its expression, with promoter deficiency leading to a substantial reduction in Maoa activity in various human cell lines (Gupta et al., 2015). Methylation mechanisms can regulate MAOA-associated long noncoding RNAs (MAALIN), which, in turn, inhibit MAOA expression in the brain (Labonté et al., 2021). Additionally, Hdac6, a key player in misfolded protein degradation, can mediate the transport of misfolded proteins for aggregate degradation (Liu et al., 2016). The excessive translation speed caused by the GAC best codon may result in protein misfolding and subsequent Maoa degradation. PTBP3, a well-studied RNA-binding protein, is involved in pre-mRNA splicing and various aspects of mRNA metabolism, including polyadenylation, mRNA stability, and translation initiation (Romanelli et al., 2013). PTBP3 binds to Maoa’S 5’-UTR, promoting ribosome entry and translation. However, Maoa translation products are ultimately reduced due to ubiquitination-mediated degradation of RBBP6 and Hdac6.RAM22 plays a role in pre-mRNA splicing as part of activated spliceosomes, participating in the first step of pre-mRNA splicing. Zcchc8 is involved in monitoring and turnover of abnormal transcripts and noncoding RNAs. Yctd1 is a regulator of alternative splicing and recognizes N6-methyladenosine (m6A)-containing RNAs (Shima et al., 2017). These cleavage factors specifically bind to the WT sequence, ensuring mRNA activity and stability. On the other hand, the binding proteins involved in the KI sequence include RAMx2 from the RAMX family, which participates in pre-mRNA splicing but is slightly less crucial in mRNA cleavage than RAM22. Adar can alter mRNA translation by modifying amino acid sequences, splicing site recognition sequences, and RNA sequences involved in nuclease recognition. SLTM, when overexpressed, acts as a general transcription inhibitor, ultimately leading to apoptosis. These differences in binding proteins contribute to lower mRNA levels and reduced Maoa product levels in the KI sequences, which align with the findings from QPCR and WB experiments.

While DNA levels in each cell remain relatively constant, mRNA levels are subject to dynamic fluctuations, making RNA-seq a powerful tool for quickly determining changes in transcriptional activity. To investigate whether the synonymous mutation in the Maoa gene leads to differences in the expression of genes involved in transcriptional and translational regulation, RNA-seq was conducted on RNA extracted from mouse brain regions. The QPCR verification results indicated an upregulation in the expression of genes such as Drd1 and Dbh enzyme. These findings suggest that the midbrain limbic dopaminergic system, which can enhance dopaminergic signaling from the ventral tegmental area (VTA) to the ventral striatal nucleus accumbens (NAc), may be influenced by the Maoa gene and play a role in addiction. This enhancement in dopaminergic signaling is achieved by both increasing the excitatory input of dopaminergic neurons and inhibiting GABAergic neurons in the VTA region (Lüscher, 2016).

Additionally, KEGG classification enrichment analysis revealed that the Sf3a2 gene, involved in pre-mRNA splicing as part of the splicing factor SF3A complex, was downregulated. This decrease in mRNA processing reflects the instability of the KI-type gene mRNA. Furthermore, several translation-related genes, including those involved in ribosomal subunit composition, DNA methylation, shearing factors, protein folding, sorting, and degradation, exhibited differential expression in the PFC brain region. These results demonstrate that synonymous mutations can impact the expression and stability of the Maoa gene by regulating the upward and downward regulation of genes related to transcriptional and translational processes.

Our study provides valuable insights into the influence of coding region SNP synonymous mutations on epigenetic regulation, protein synthesis dysfunction, and dopamine system abnormalities. It sheds light on the cytoplasmic function of RNA-binding protein (RBP) in Maoa translation, offering a promising avenue for further research in this area. Using CRISPR/Cas9 technology to create a genetic mouse model that simulates the expression of the Maoa c.1409 T > C mutation in animals is a commendable approach to investigate potential epigenetic effects. Animal models are crucial for understanding the in vivo implications of genetic mutations. While Our study has made significant progress, there are indeed some limitations, such as the cell model of the gene mutation. Further experiments could delve deeper into the molecular mechanisms involved in how RBP proteins regulate specific processes related to transcription and translation. This would provide a more comprehensive understanding of the underlying mechanisms at play. Overall, Our research contributes to the growing body of knowledge on the impact of synonymous mutations on gene expression and protein function, particularly in the context of the dopamine system and related disorders. It also highlights the potential for future investigations in this field.

The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found at: https://www.ncbi.nlm.nih.gov/genbank/, GSE237077.

The animal study was approved by the China National Tobacco Quality Supervision and Test Center, CTQTCSYXK- 2023003. The study was conducted in accordance with the local legislation and institutional requirements.

KL: Data curation, Methodology, Writing – original draft. LF: Formal analysis, Validation, Visualization, Writing – original draft. HW: Supervision, Writing – review & editing. YT: Data curation, Writing – original draft. SZ: Writing – review & editing. QH: Supervision, Funding acquisition, Writing – original draft. FL: Writing – review & editing. HC: Funding acquisition, Writing – review & editing. HH: Data curation, Funding acquisition, Supervision, Writing – review & editing.