Epitranscriptome analysis: The hidden secret control of RNA modification
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Epitranscriptome analysis: The hidden secret control of RNA modification
Epitranscriptome analysis: The hidden secret control of RNA modification. Epitranscriptomics has received more and more attention from scientists and has become one of the hot fields that have recently emerged. So far, more than 170 chemical modifications have been found on RNA .
On the last day of 2016, Nature Methods rushed to release the 2016 annual technology. Many people think that the annual technology of the year must be the CRISPR technology of the fire, but it is not common. For the forward-looking annual technology inventory, one is not common. Noun: Epitranscriptome analysis is the correct solution.
The name Epitranscriptome analysis is prefixed by the Greek “epi”, which refers to any modification added to nucleotides except for known function or heredity. For decades, scientists have hardly noticed RNA modification, because markers on RNA were discovered in the 1960s and 1970s, but everyone only paid attention to tRNA and rRNA, as well as epigenetics on DNA. Retouch.
But as scientists discovered chemical markers that appear in all types of RNA, the “Reader Writer” and “Eraser Eraser” that dynamically add or remove these markers have rekindled interest in RNA modification. For example, the association between an enzyme that removes a methyl group from adenine and the risk of Alzheimer’s disease suggests that this modification plays an important regulatory role in neurological health.
As a result, epitranscriptomics has received more and more attention from scientists and has become one of the hot fields that have recently emerged. So far, more than 170 chemical modifications have been found on RNA . These modifications are largely distributed on non-coding RNA (ncRNA), especially rRNA, tRNA and snRNA, and are necessary for ncRNA to function normally in translation and splicing. Excitingly, the researchers found that m6A (N6-methyladenosine), m1A (N1-methyladenosine), m5C (5-methylcytidine), hm5C (5-hydroxylmethylcytidine), I (inosine) and ψ (pseudouridine) and other chemical modifications also It is distributed on eukaryotic mRNA and affects the metabolism and function of mRNA. Especially with the new discoveries of many mRNA modification enzymes (Writer), demodification enzymes (Eraser) and modified recognition proteins (Reader), the reversible changes and dynamic regulation of mRNA chemical modification have renewed the interest of researchers.
Epitranscriptomics research scope
Epitranscriptomics (epitranscriptomics, also known as “RNA epigenetics”) refers to post-transcriptional RNA modifications that bring functionally related changes to the transcriptome. Epitranscriptome modification includes several important RNA processing events, including RNA editing, methylation, and splicing (Figure 2).
In terms of whether it encodes a protein, RNA can be divided into coding RNA (coding RNA) modification and non-coding RNA (non-coding RNA, ncRNA). The former refers to mRNA, and the latter includes many types, such as the well-known tRNA and rRNA, and snoRNA involved in RNA modification.
The most researched mRNA modification is m6A modification. As early as the 1970s, scientists discovered m6A modification in RNA. Then more and more studies have proved the importance of m6A modification: m6A modification and mRNA stability, splicing Processing, translation, and processing of microRNAs; m6A is also related to stem cell fate and biological rhythms, which can promote stem cells from self-renewal to cell differentiation. Researchers have found that methylation shortens the half-life of mRNA and reduces its abundance. It can be said that m6A modification affects almost every step of RNA metabolism.
In recent years, with the deepening of research, a lot of research work has shifted from mRNA to focus on the important role of non-coding RNA methylation in the occurrence and development of diseases. It has been found that m6A methylation of non-coding RNA can contribute to stem cell differentiation, It plays a key role in the proliferation of cancer cells. Like mRNA, there are many chemical modifications on lncRNA. For lncRNA, m6A methylation can regulate the secondary structure of lncRNA, lncRNA binding protein, and the ceRNA mechanism of lncRNA, as well as the m6A modification of target genes.
In addition, there are m6A modifications on circular RNA, which can affect the interaction between circRNA and RNA binding protein (RBP), and mark endogenous RNA to distinguish it from exogenous RNA and avoid recognition and attack by the self-immune system .
Small RNA, such as microRNA, tRNA-derived small RNA (tsRNA, including tRF&tiRNA), etc., have a variety of different modifications on the RNA molecule. These modifications can regulate the activity of small RNAs on the one hand, and can also give them new functions on the other hand. . It is known that these RNA modifications function through a variety of molecular mechanisms. For example, RNA modifications can change the targeting of miRNAs or change the affinity of tsRNA (tRF&tiRNA) with RNA-binding proteins, thereby exerting biological activity. Small RNA modification profile analysis is a new frontier of epitranscriptomics research, which has important scientific significance and clinical value.
The most important achievement in the field of RNA modification
Regarding the most important achievement in the field of RNA modification so far, Professor Samie R. Jaffrey of Cornell University believes that “the first method for mapping the m6A whole transcriptome may be the most important event in the field of epitranscriptomics. It is replicated for several other nucleotide modifications.
Before the whole transcriptome is mapped, the modified nucleotides in the hydrolyzed RNA are detected by mass spectrometry or other analytical measurements, and the modified nucleotides are found. These methods are ambiguous , Especially for low-abundance modifications: even if you have a high-purity mRNA preparation, you are still worried that trace amounts of contaminating transfer RNA (tRNA) or ribosomal RNA (rRNA) may be the source of the modification.
This makes it difficult to determine the modification Does the nucleotide come from mRNA.”
Professor Gideon Rechavi of Tel Aviv University School of Medicine in Israel said, “In my opinion, the main achievement in this field is the discovery of a new, complex, highly sensitive, and adjustable gene expression regulation layer through mRNA modification. This The new regulatory layer takes advantage of the unique characteristics of mRNA—that is, it is short-lived, highly structured, moves between cell compartments and is amplified by transcription. These effects are partly mediated by “readers”, such as The identification of methyl-specific binding proteins is a milestone in the field of modification.
The regulation of gene expression has also been adjusted through the interaction between the modification, installation and deletion of “writer” and “eraser”. In the past ten years Several important lessons emerged. First, mRNA modification is very common, thousands of gene transcripts are modified. Interestingly, some modifications are concentrated in specific transcription positions; for example, inosine is mainly found in repeated Alu sequences.
In the column, m6A preferentially modifies the m1A cluster near the stop codon and internal exons, and around the AUG start codon, indicating that each modification acts in a different mode. In addition, some modifications, such as m6A and m1A , Showing a high degree of conservation between humans and mice.
Another important achievement is the discovery that specific modifications can pass through different behavior patterns, through different readings, and depend on upstream and downstream. Another important finding is the dynamic properties of some mRNA modifications, which can quickly respond to environmental stimuli; m6A and m1A have demonstrated this dynamic property. The core role of mRNA modification is reflected in the destructive effects of abnormal modification on early development of humans and mice, as well as human cancer, inflammation, and neurodegeneration, which further emphasizes the importance of this regulatory layer. “
Professor Tsutomu Suzuki of the University of Tokyo said, “Previous biochemical research on RNA modification has focused on classic non-coding RNAs, including tRNA, rRNA, and small nuclear RNA (snRNA), because these RNAs are abundant in cells. However, recently, Whole-transcriptome analysis of RNA modifications using NGS technology has identified several base modifications, including inosine (I), m6A, m5C, Ψ and m1A in mRNA, and long non-coding RNA. This in turn has greatly broadened this. The concept of epitranscriptome is introduced.
In the past ten years, through reverse genetics and mass spectrometry, scientists have identified RNA-modifying enzymes buried in the genome of model organisms. Using this method, we successfully identified 40 RNA-modifying genes.
It is also worth mentioning that disease-related exome sequencing helps to analyze how mutations in RNA-modifying enzymes cause many human diseases. “
How to study RNA modification
Judging from the current research results, the apparent transcriptome has far-reaching influence as a means of superimposing plasticity on other genomes and connecting transcriptomes. The “code” of the epitranscriptome can not only enable or enhance specific chemical reactions in RNA catalysis or RNA-dependent reactions, but also change the structure-function relationship of RNA, thereby providing an additional layer of gene regulation in a spatio-temporal and signal-dependent manner.
However, the functional study of the epitranscriptome lags behind that of the epigenome because of the lack of sensitive and robust technology that can detect these epitranscriptome markers in the transcriptome range. There are several major challenges in studying the epitranscriptome. First, most RNA modifications cannot be directly detected by high-throughput sequencing.
Because chemical modifications to RNA usually do not change the base-pairing properties of modified bases, reverse transcription (RT) will simply erase these modifications and make them indistinguishable from regular RNA bases. Second, although rRNA, tRNA, and snRNA are abundant, other types of RNA, such as mRNA and long non-coding RNA (lncRNA), may be less abundant. Third, there is a lack of existing computing tools to facilitate the ability to identify modified sites from sequencing data.
Fortunately, in recent years, significant progress has been made in research methods for different epitranscriptomes. These new tools help researchers determine the location of RNA modifications and reveal the different distribution patterns of these modifications throughout the transcriptome. When these methods are combined with other emerging tools (such as genome editing tools), the targets of RNA-modifying enzymes have been determined. In addition, these techniques also reveal the dynamic nature of different epitranscriptome markers under different physiological conditions. At the same time, these tools enable people to discover “reader” proteins that selectively recognize specific epitranscriptome markers and determine their functions. Therefore, new research tools can not only perform a comprehensive analysis of the epitranscriptome, but also a valuable resource for functional epitranscriptome research .
⑴ Detection and analysis method
Methods for studying RNA modification include liquid chromatography (LC-MS), high-throughput sequencing-based methods, and chip analysis.
① mRNA&lncRNA epitranscriptome chip
The potential function of RNA modification depends not only on which gene transcript it modifies, but also on the percentage of the modified part in the transcript. However, most current RNA modification detection methods at the transcriptome level focus on finding modified sites on transcripts, and cannot quantitatively detect the percentage of modified transcripts. The lack of this type of quantitative information has attracted the attention of more and more researchers.
The potential impact of mRNA modification depends not only on its molecular effects, but also on the percentage of modified transcripts. For example, a modification that can accelerate the degradation of mRNA. If only 1% of the transcript is modified, it is obviously unlikely to produce any biological function. However, when a modification can promote the translation of mRNA into a new protein subtype, even the modification level Very low, may also produce important biological functions. The limitation of current m6A and Ψ detection methods is the lack of quantitative information on the degree of modification. The regulatory effect of m6A can be inferred by the method of pulldown m6A to detect the relative enrichment of specific sequences in different states, but the absolute amount of modified mRNA cannot be obtained from these data. The new high-throughput detection method that can quantify m6A and Ψ will greatly promote the development of this field .
Another important issue is to clarify the dynamic changes in the stoichiometry of RNA modification. At present, the focus of epitranscriptomics research is mostly which sites are modified, rather than the proportion of each modified site in RNA. Low-throughput analysis of m6A modification sites of mRNA and viral RNA shows that the proportion of any m6A modification sites will not reach 100%. The stoichiometric change of the modification may be a dynamic parameter of RNA biological modification. Modifications can affect the structure of mRNA and/or the recruitment of RBPs. Modification of any particular site will cause the same mRNA population to be divided into two mRNA subgroups only due to differences in structure or binding readers. Therefore, changing the stoichiometric number of modifications may be a mechanism for the same RNA transcript to perform different functions. There is an urgent need for a high-throughput method that can detect modified stoichiometry to clarify the problem of epitranscriptomics in this respect .
Arraystar mRNA&lncRNA apparent transcriptome chip combines dual fluorescence channel chip technology and RNA modification immunoprecipitation technology to quantitatively detect RNA modification at the transcript level. A quantitative epitranscriptome map can provide important information for the study of RNA modification regulation.
②Liquid chromatography-mass spectrometry
LC-MS is a powerful technique that can detect modified RNA nucleosides with excellent sensitivity and high specificity. However, in the context of measuring mRNA modifications, there are two key limitations.
First, the ability of LC-MS to perform site-specific detection is limited. In the context of mRNA, so far, MS has only been applied to fully digested nucleosides, so it can estimate the “overall” level of the modification in the sample, but excludes the possibility of assigning the modification to a single site. When applied to partially digested RNA oligonucleotides, LC-MS can also provide site-specific information in principle. However, this analysis usually requires tens to hundreds of nanograms of at least partially purified molecules, which is impractical for mRNA because of its high heterogeneity and low abundance characteristics.
Second, compared with tRNA and rRNA, the relative level of modification in mRNA is lower, so interpreting LC-MS results becomes more and more difficult. Even low-level contamination from highly expressed tRNA and rRNA (which can never be completely avoided, although they can be estimated to some extent based on tRNA-specific or rRNA-specific nucleosides) can also lead to an overestimation of the level of mRNA modification. m6A is the first mRNA modification analysis conducted extensively by MS. There are fewer restrictions in this regard because it is highly abundant in mRNA but not present in tRNA and only exists at two sites in rRNA (hence , Pollution may lead to underestimation of the actual m6A level).
In contrast, most non-m6A epitranscriptomes are very rare in mRNA, and more common in tRNA and rRNA1, which severely limits LC-MS’s ability to understand its abundance and dynamics. In some cases, the abundance of modifications estimated by LC-MS (based on that the modification is considered to be present at a high level in mRNA) and genomic-based methods (generally unable to detect a large number of modifications in mRNA); therefore, the source of these differences may be TRNA or rRNA contaminants in the mRNA part. Another possibility is that certain modifications are the result of RNA damage, which may lead to alkylation and oxidation of nucleotides. This non-enzymatic modification randomly scattered throughout the transcriptome can be detected by LC-MS, but not by genomic methods, which requires accumulation of signals at specific sites.
③Methods based on sequencing
In the past decade, analysis of various modifications based on high-throughput sequencing has become a key driving force for progress in this field. These methods can inform the existence of the modification in the mRNA and its precise location in the transcriptome.
Compared with LC-MS, in principle, a single workflow can know the abundance of all modifications, and the use of genomic methods to identify modifications requires the development of a dedicated and unique workflow for each modification. The fundamental challenge that this method needs to overcome is that most modifications are “invisible” in standard sequencing, that is, they will not leave any traces after reverse transcription of RNA into cDNA, which is based on Illumina standards The prerequisite step method for sequencing. Therefore, scientists have developed a set of methods to present different visible modifications. For m6A, the main detection method relies on anti-m6A antibodies, which are used to selectively immunoprecipitate short RNA fragments containing m6A.
Although immunoprecipitation is a universal method that can in principle be used to detect any modification, in fact, antibody-based methods have limited utility in mapping the non-m6A epitranscriptome. Once researchers tried to use it to map m1A or ac4C36, it is now believed to cause a large number of false positive sites, which may be caused by antibody cross-reactions. Anti-m6A antibodies also have this cross-reactivity; however, given the relatively high abundance of m6A, the signal-to-noise ratio is still controllable. In contrast, the presence of modifications at a lower order of magnitude abundance resulted in a dramatic increase in the ratio of non-specific to specific binding events.
Therefore, for the vast majority of non-m6A epitranscriptomes, methods have been developed that utilize the unique chemical properties of modified bases to make them visible after reverse transcription. This is achieved through different strategies, for example, by changing the modification-specific chemical substances that modify the identity of the base or by specifically linking a large number of residues to the modified base to cause premature truncation during reverse transcription (see the table below). These experimental protocols usually lead to mismatches or deletions of modified sites, or to “stacking” of reads, which selectively start or end at a specific position, and then the existence of the modification can be inferred from it. The main advantages of such chemical genomics methods are their excellent, usually single-nucleotide resolution, and their ability to provide relative quantification of modification levels, sometimes even absolute quantification .
For example, Kangcheng Biotechnology has established a highly sensitive and absolute quantitative real-time fluorescent quantitative PCR quantitative analysis method, which can accurately detect m6A modification sites in RNA at a single nucleotide resolution level and perform quantitative analysis on unknown templates. This method can be successfully applied to the accurate detection of m6A modification in actual biological samples, even samples with low abundance RNA.
In recent years, some new sequencing technologies have also appeared one after another. For example, in 2017, the research team of the Peking University Yichengqi Research Group and the Weizmann Institute of Israel independently reported a new method for detecting m1A at the single-base resolution level-m1A. -MAP and m1A-Seq. In 2019, Bryan C. University of Chicago The Dickinson team worked with He Chuan’s team to research and develop an evolutionary platform that can quickly select reverse transcriptases, and use this platform to study m1A modifications on mRNA to achieve single-base level m1A detection.
In addition, direct RNA nanopore sequencing has also attracted much attention. Christopher Mason of Cornell University, a pioneer in RNA epigenetics, said, “In the past, we usually used antibodies or chemical analysis to infer the modification status of RNA. Recently, we have only started to perform direct RNA sequencing. Using a nanopore sequencer, we can directly sequence RNA without the need for reverse transcription. For the first time, we directly measure RNA modifications: what are the modifications in the entire molecule and what isomers are there .”
Therefore, it can be said that nanopore sequencing can complete sequencing, read the m6A, m5C modification of RNA sequence (usually reading single molecule sequence is delayed compared with unmodified base), and the read length advantage makes it suitable for variable shearing research and The isomer has more advantages, it does not need to be spliced, and it is not easy to lose rare molecules, but it is more expensive.
In a recent hot article “The Architecture of SARS-CoV-2 Transcriptome SARS-CoV-2”, the researchers used the MinION nanopore sequencer to perform DRS sequencing and obtained 879,679 reads (1.9 Gb). The result It not only proves that viral transcripts dominate, but also nanopore DRS RNA-based single-molecule detection provides a unique opportunity to examine the multiple transcriptome characteristics of a single RNA molecule and analyze the characteristics of new coronavirus RNA modification .
⑵Currently known RNA epigenetic transcription modification:
m6A is the most abundant chemical modification on eukaryotic mRNA. It is catalyzed by methyltransferase complexes (including METL3, METL14, WTAP, KIAA1429, RBM15, RBM15B) and can be removed by the demethylase ALKBH5 or FTO. A variety of proteins or complexes that specifically recognize the m6A site have been discovered, including YTH family proteins (YTHDF1-3, YTHDC1), transcription initiation complex eIF3, ribonucleoprotein (HNRNPA2B1, HNRNPC), and RNA binding protein SRSF2 .
m6A is mainly distributed in the vicinity of the stop codon and the 3’UTR region, affecting RNA pairing, altering RNA secondary structure, or directly recognized by proteins, thereby regulating mRNA maturation, alternative splicing, stability and translation processes. Compared with the A-U pairing, the m6A-U pairing is less stable, which triggers the unwinding of the double-stranded RNA and the transformation of the secondary structure. m6A often stacks in the transition region between double-stranded and single-stranded, enhancing the stability of the conformation after RNA transformation. Demethylation can restore the original conformation of mRNA.
This conformational shift may lead to changes in the interaction between mRNA and different proteins, resulting in different biological effects. m6A can be directly recognized by the hydrophobic domain of a specific protein. For example, YTH family proteins can specifically recognize m6A, especially the conserved sequence of GGm6ACU. One of its members, YTHDC1, recognizes and binds to m6A, and regulates alternative splicing of targeted mRNA. After the other member YRHDF2 binds to m6A, it recruits the CCR4-NOT complex to promote the degradation of targeted RNA.
In UV radiation or heat shock reaction, the transcription initiation complex eIF3 binds to m6A in the 5’UTR region to promote the cap-independent translation process. The m6A in the coding region can be recognized by SRSF2 and is involved in the regulation of adipogenesis. In Drosophila, YTHDC1 homologous protein recognizes the m6A site on sex-lethal mRNA and regulates its alternative splicing, thereby controlling the sex of Drosophila. As mentioned earlier, m6A-mediated regulation of mRNA stability is also very important for stem cell differentiation and biological rhythm clock control. In addition, m6A can also affect translation elongation by affecting the rate and fidelity of the pairing of mRNA and tRNA anticodons.
m1A is a newly discovered reversible apparent transcription modification, which can be removed by the RNA repair enzyme ALKBH3. So far, no clear m1A modification enzyme and modification recognition protein have been found. Unlike m6A, m1A has a low expression abundance, mainly distributed in the 5’UTR region of mRNA, and may be involved in the process of regulating translation initiation.
m1A can completely prevent Watson-Crick pairing, cause RNA double-strand unwinding, and promote the electrostatic interaction of RNA-protein or the formation of RNA variable secondary structure. The biological function of m1A is still unknown. Studies have found that under stress conditions such as heat shock or nutrient deprivation, the increase in m1A expression levels in cells may be involved in the cell’s stress response by promoting cap-dependent translation.
m5C and hm5C
m5C is widely distributed on tRNA and rRNA, and has the functions of stabilizing the secondary structure of tRNA, influencing the conformation of the anticodon loop, and maintaining the fidelity of rRNA translation. Recent RNA sequencing results found that there are more than 8,000 m5C sites in the coding and non-coding regions of mRNA, and a considerable number of sites are concentrated in the 5’UTR and 3’UTR regions. m5C can be catalyzed by methyltransferase NSUN2 or TRDMT1 and oxidized by dioxygenase TET to form hm5C.
hm5C may undergo further oxidation to form f5C, which then turns back to cytosine (C). m5C does not affect base pairing, but may enhance base stacking and the hydrophobic interaction between RNA and protein. m5C has a variety of biological functions. After p16 mRNA is modified by NSUN2 enzyme with m5C, its degradation is inhibited and its stability is enhanced.
During the cell cycle, the expression of NSUN2 is precisely regulated. NSUN2 can add m5C modification in the 3’UTR region of CDK1 to promote its translation; meanwhile, add m5C modification in the 5’UTR region of CDKN1B to inhibit the translation of CDKN1B. The two work together to enhance cell proliferation.
m5C is also related to the translation control of aging-related genes. Overexpression of NSUN2 can delay the occurrence of replicative senescence. As the oxidation product of m5C, hm5C can also enhance translation efficiency. hm5C is highly expressed in the brain of Drosophila and may be involved in the development of the brain of Drosophila.
Pseudouridine ψ, often referred to as the fifth type of nucleotide, is formed by isomerization of uridine (U). In the mRNA of human cells and mouse tissues, the ratio of ψ/U is about 0.2-0.6%. The isomerization reaction of uracil and pseudouracil is catalyzed by PUS enzyme alone or together with H/ACA ribonucleoprotein. Pseudouracil can reduce the variability of RNA conformation, enhance the stability of base pairing and the polar interaction with protein. Pseudouracil may regulate mRNA stability and gene expression, and participate in the heat shock response of yeast, but the specific mechanism is currently unclear.
Inosine modification, often called A-to-I editing, is the most common way of RNA editing in higher eukaryotes, and it is done by adenylate deaminase ADAR. A-to-I editing mainly occurs in Alu elements in non-coding regions or intron regions. A-to-I editing completely changed the base pairing characteristics, AU pairing was converted to IC pairing, thereby changing the encoded amino acid. For example, A-to-I editing recodes glutamine in the brain’s glutamate receptors into arginine, which leads to changes in calcium ion permeability. In addition, A-to-I editing can also change alternative splicing, regulate the production and function of miRNA, and monitor innate immune responses.
In 2018, Professor Shalini Oberdoerffer from NIH in the United States published a research paper in Cell: Acetylation of Cytidine in mRNA Promotes Translation Efficiency, revealing for the first time that there are a large number of ac4C modifications on mRNA, and ac4C affects mRNA stability and translation efficiency.
N4-acetylcytidine (ac4C), N4 acetylcytosine, is a conservative chemical modification in eukaryotic prokaryotes. Early studies suggested that ac4C mainly exists on tRNA and 18S rRNA. Recent studies have shown that a large amount of ac4C is also present on mRNA, and its abundance is not even lower than the m7G cap modification carried by mRNA. NAT10 is the only protein identified so far that has both an acetylase domain and an RNA binding domain, so it is considered to be an RNA ac4C modifying enzyme.
As a result, acetylation, as a new type of mRNA modification, extends the apparent transcriptome beyond the known types of methylation and isomerization. These works have also expanded the known library of mRNA modifications and determined the existence of a variety of chemical modifications. New models of mRNA modification are likely to continue to emerge in the future. A further understanding of the functions of these modifications, the pathways and mechanisms that regulate their presence on specific transcripts, and their functions in various physiological and disease contexts will help to understand the epitranscriptome in gene expression more comprehensively The role of .
The 5′-end hypermethylated TMG modification is one of the first modifications discovered. In many RNAs transcribed by RNA polymerase II, especially non-coding RNAs, such as snRNA, an important component of spliceosome, the m7G methyl cap at the 5’end will be hypermethylated to form N2, N2 with three methyl groups. , 7-Trimethylguanosine (TMG) caps are widely found in eukaryotes.
Trimethylguanosine synthase (Tgs1) has been proven to be the only methylase that synthesizes TMG caps in many organisms, and it is evolutionarily conserved from yeast to humans. Due to the limitations of in vitro experiments, the regulatory mechanism of TMG modification on spliceosome RNA is still unclear. In most multicellular model organisms, TMG modification cannot be directly studied in vivo.
Therefore, TMG modification has been It has been discovered for more than 50 years, but its function in multicellular organisms is not clear.
(source:internet, reference only)
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