N1-methyladenosine formation, gene regulation, biological functions, and clinical relevance

Summary

N1-methyladenosine (m1A) is an adenosine moiety whose N1-position is methylated. m1A methylation is a prevalent, abundant, and conserved internal post-transcriptional modification among prokaryotic and eukaryotic RNAs, especially in higher eukaryotic cells. Numerous studies have revealed that m1A methylation plays a critical role in the biogenesis of various RNAs, thereby regulating different biological functions and pathogenesis. In this review, we systematically and comprehensively summarize the installation, removal, and recognition of m1A and highlight the critical effects of m1A on the post-transcriptional metabolism of various RNAs. We emphasize the importance of m1A both in the growth of organisms and pathogenesis of various diseases, particularly cancers. Finally, we also focused on the fact that excretion of m1A in human urine is strongly associated with the progression of a variety of diseases, and we suggest that m1A levels in urine can be quantified for early diagnosis of some diseases as well as for monitoring during disease evolution.

Graphical abstract

m1A methylation plays a critical role in the biogenesis of various RNAs. In this review, Xiong et al. systematically and comprehensively summarize the installation, removal, and recognition of m1A and emphasize the importance of m1A both in the growth of organisms and pathogenesis of various diseases, particularly cancers.

Introduction

RNA methylation is an important post-transcriptional epigenetic modification that plays an important role in various biological processes, such as gene expression, genome editing, and cell differentiation.^1,2 With the rapid development of specific antibodies and high-throughput sequencing technologies, more than 160 chemical modifications that exist on various RNA molecules have been identified. These mostly occur at the inside or at both ends of the RNA molecular chains, and most of them more commonly occur on tRNA and rRNA.³ Subsequent studies have revealed that various forms of covalent modifications occur on mRNAs, including N1-methyladenosine (m1A, Figure 1A), N6-methyladenosine (m6A), N7-methylguanosine (m7G), 5-methylcytidine (m5C), N4-acetylcysteine (ac4C), 5-formylcytidine (f5C), 2′-O-methylated nucleoside (Nm), and pseudouridine (ψ) modifications.⁴ In addition to the m6A methylation, m1A methylation is the second most prevalent, abundant, and conserved internal post-transcriptional modification in eukaryotic RNAs, and plays an important regulatory role in post-transcriptional RNA modifications, such as RNA assembly, splicing, export, transport, translation, stabilization, and degradation.^5,6

Figure 1.

m1A modifications in tRNAs

(A) m1A and m1I nucleosides. (B) All identified m1A modification sites in tRNAs (in red). The letters A refer to Archaea, B to bacteria, and E to Eukarya, which are the three organisms, while M refers to mitochondria. ALKBH1-mediated f5C34 formation is required for normal mitochondrial function (in green). ∗m1A9 is mainly found in mt-tRNA and only a few in eukaryotic cytoplasmic tRNA.

The investigation of the distribution and function of m1A methylation has aided in the rapid development of RNA epigenetics and epitranscriptomics. Different methylases perform m1A methylation, which occurs at different sites of tRNA, rRNA, and mRNA, using the methyl moiety provided by S-adenosylmethionine (SAM).^7,8 tRNA m1A58 is widely conserved in bacteria, Archaea, and eukaryotes, especially in all initiation tRNAs (tRNA_i^Met), which play a key role in tRNA stability and translation initiation.^9,10 m1A methylation in rRNA mainly occurs on the large subunit of the ribosome, which has been shown to affect ribosome biogenesis and mediate cellular resistance to antibiotics (e.g., anisomycin) and sensitivity to specific stresses (e.g., oxidative stress).^11,12 So far, no m1A methylase that specializes in mRNA m1A methylation has been identified, and most m1A sites are within the 5′ untranslated regions (UTRs) in nuclear-encoded mRNAs, where they play an important role in maintaining protein translation. In contrast, in mitochondria-encoded mRNA (mt-mRNA), m1A sites are mainly located in the protein-coding sequence (CDS), and, thus, are involved in the inhibition of the translation process of mitochondrial proteins.¹³

m1A methylation and its related regulators play various important roles in development and diseases, including regulation of growth and development,^14,15 stress responses,^16,17 and tumorigenesis.^18,19,20 For instance, nucleomethylin (NML)—which is responsible for catalyzing the formation of m1A in rRNA—and its deletion lead to severe impairment of erythropoiesis during embryonic development.¹⁴ Moreover, m1A and m7G in tRNA have been found to be involved in the stress responses of plants to environmental stressors.¹⁶ In addition, m1A methylation was found to be significantly increased in the urine of patients with cancer.^18,19,20

Considering the complexity and importance of m1A, some researchers have reviewed the recent advances in the distribution, formation, and function of m1A in tRNA.^21,22,23 However, a comprehensive description of the distribution and function of m1A in the three types of RNA—tRNA, rRNA, and mRNA—and the specific roles of m1A-related regulators in the formation, erasure, and recognition of m1A are still lacking. In this review, we comprehensively summarize the discovery and distribution of m1A in various RNAs, m1A methylation modification-related enzymes, the functional role of m1A, and its role in growth and development, stress responses, and various human diseases, especially cancer.

m1A methylation in various RNAs

Shortly after the discovery of the base N1-methyladenine in 1961,²⁴ Dunn et al.²⁵ isolated N1-methyladenosine mononucleotide from RNA. After decades of continuous exploration, researchers have successively identified that m1A sites are also present in tRNA,^9,21,26,27 rRNA,^7,12,28 mRNA,^7,13,29,30 long non-coding RNA (lncRNA),^13,29,31 and mitochondrial transcripts (Figure 2),^12,13,30,32 with the highest abundance in tRNA and rRNA. In contrast, the level of m1A in mRNA is relatively low, but it is still one of the most abundant modifications in mRNA in addition to m6A.

Figure 2.

Timeline of m1A-related research on various RNAs

m1A on tRNAs

Compared with those in other RNAs, tRNAs possess the most chemical modifications and have the widest chemical diversity. Eukaryotic tRNAs contain, on average, 13 modifications per molecule, ranging from base isomerization to base and ribose methylation.³³ Chemical modifications in tRNAs help improve the translational efficiency and fidelity as well as help in the correct folding of the tRNA-specific “cloverleaf” or inverted L-shape structure.

In 1966, m1A was first discovered in yeast phenylalanine tRNA (tRNA^Phe) by Bhandary et al.²⁷ Nearly 40 years later, Sprinzl et al.²⁶ counted 564 tRNAs from bacteria, Archaea, and eukaryotes and found that 264 of them contained m1A modification sites, which appeared on adenosine at positions 9, 14, 22, and 58 of the tRNA sequence (Figure 1B). Among them, the m1A modification at position 57 is the intermediate in m1I57 (N1-methylinosine modification at position 57) biosynthesis in archaeal tRNAs and is only transiently present (Figure 1A).^34,35 m1A modifications at positions 14 and 22 (m1A14 and m1A22) are considerably rare. m1A14 has only been reported in several mammalian cytoplasmic tRNA^Phe,²⁶ while m1A22 has been found only in tRNA^Ser and tRNA^Tyr from Mycoplasma capricolum³⁶ and Bacillus subtilis.^37,38 In addition, m1A14 has not been observed in human-derived cellular tRNAs.¹³ m1A58 is located on the TψC loop of tRNAs, which is extremely conserved in bacteria, Archaea, and eukaryotes and plays a crucial role in tRNA stability.⁹ To date, m1A58 has been observed in all sequenced eukaryotic initiator tRNAs.²¹ At position 9 of human tRNAs, m1G is prevalent in cytoplasmic tRNAs, whereas m1A is more common in mitochondrial tRNAs.^39,40 Therefore, m1A modifications at positions 9 and 58 (m1A9 and m1A58) of tRNA have been the research hotspot.

In addition to nuclear-encoded transcripts, m1A modifications are also ubiquitous in human mitochondrial RNA. Fourteen of the 22 tRNAs encoded by mitochondria bearing an adenosine residue at position 9 are m1A modified.¹³ m1A modifications are required for the function and structure of tRNAs.^41,42

m1A on rRNA

A substantial amount of modified nucleotides are also observed in rRNAs. In contrast to the 25 different types of modifications observed in tRNAs of budding yeast, only 12 distinct types were discovered in rRNA, which include Nm, ψ, m1A, m7G, ac4C, and m5C, with Nm and ψ modifications being the dominant modifications.¹¹

In Saccharomyces cerevisiae, the 25S rRNA contains two m1A modification sites, at positions 645 and 2,142.²⁸ The modification at position 645 is transmethylated by ribosomal RNA processing 8 (RRP8) to form m1A,^43,44 while base methyltransferase of 25S RNA (BMT2) is involved in the base modification at position 2,142.²⁸ In 2016, Dominissini et al.⁷ uncovered the presence of m1A modifications for the first time at position 1,135 of the cytoplasmic 28S rRNA in mouse-derived cells. In addition to nuclear-encoded rRNA, m1A modifications were also observed in two mitochondrial rRNAs. m1A was present at position 947 of the mitochondrial 16S rRNA (16S mt-rRNA), which is highly conserved across vertebrates.³² In subsequent studies, m1A was also found to be present in the 12S rRNA encoded by human mitochondria (12S mt-rRNA). Unlike m1A947 in 16S mt-rRNA and m1A1322 in 28S rRNA, which are both highly modified, the newly identified m1A sites in 12S mt-rRNA have a relatively low modification level.¹³ m1A of bacterial 16S rRNA at position 1,408 enhances bacterial resistance to aminoglycosides.¹²

m1A on mRNA

Previous studies on m1A have focused on tRNA and rRNA. Recently, a large number of m1A modifications have been found on both nuclear-encoded and mitochondrial-encoded mRNAs in mammalian cells. In 2016, Li et al.²⁹ first reported that m1A is a more prevalent modification in mRNAs, existing in 887 transcripts of 600 human genes. The m1A/A ratio of mRNAs isolated from various human cell lines was approximately 0.02%, and this m1A level was approximately 5%–10% of the m6A level. In HEK293T cells, m1A was found to be mainly enriched within the 5′ UTRs and near start codons (50-nucleotide window centered on the start codon), and the level of m1A methylation gradually decreased from the 5′ end to the 3′ end of mRNA.^7,29 Among them, the m1A modification located in the CDS is more likely to occur on the codon of arginine (CGA), whereas no m1A modification was detected for the AUG start codon. Interestingly, 22 m1A sites were observed at the first nucleotide (cap + 1) at the 5′ end of the mRNA.¹³ m1A modifications were also found to be present in 10 of 13 mt-mRNAs, and these m1A sites were almost always within the CDS region, with a preference for adenosine at the third position of the codon,¹³ which is different from the distribution of m1A in nuclear-encoded mRNAs. This could be due to the very tight gene arrangement of the mitochondrial genome, with very short gene spacer regions, and the transcriptome containing no or only short UTRs. The modification level of m1A in mt-ND5 was the highest among all mt-mRNAs.^13,30

m1A writers, erasers, and readers

Recently, it was found that three types of molecules are usually involved in the modification process of m1A methylation, namely writers, erasers, and readers, which are collectively referred to as RNA-modifying proteins. Writers are responsible for the methylation of RNA, erasers play a role in removing the m1A from RNA, and readers can recognize and bind to the m1A-modified transcript and participate in the regulation of downstream biological processes. m1A is involved in almost all processes of RNA metabolism, including RNA stability, splicing, folding, export, transport, and protein translation efficiency. It plays an important regulatory role in cell development, disease progression, and other biological processes.^{9,13,32,45,46,47,48,49}

Several investigations have revealed that the m1A modification mainly occurs in the nucleus, whereas its functional execution occurs in the cytoplasm. Consistent with this concept, m1A writers (TRMT6/TRMT61A, NML, and BMT2)^10,28,44,50 and erasers (ALKBH1, ALKBH3, and FTO)^29,51 mostly exist in the nucleus and perform methylation or demethylation of RNA; in contrast, most readers (YTHDF1, YTHDF2, and YTHDF3) exist in the cytoplasm and are related to the translation and degradation of m1A-modified RNA.^47,48,49

Writers

Given that m1A was originally discovered in tRNA and the importance of tRNA m1A58, researchers have been striving to identify the key enzyme that enables m1A modification of tRNA. In 1977, tRNA m1A58 methyltransferase (MTase) was first purified from rat liver, which exhibited m1A MTase activity against Escherichia coli tRNA₂^Glu in vitro.⁵² A decade later, Anderson et al.¹⁰ identified the genes encoding tRNA m1A58 Mtase in S. cerevisiae, which are encoded by both tRNA Mtase 6 (TRMT6) and tRNA Mtase 61A (TRMT61A) together. Up to now, six m1A methyltransferases have been detected in human cells: TRMT6/TRMT61A, TRMT61B (localized in mitochondria), TRMT10C (localized in mitochondria), NML (including homologs RRP8, RRAM-1⁵³), BMT2, and MTR1. The first three are mainly responsible for tRNAs methylation (Table 1, Figure 3). Methionine was labeled with deuterated elements (D₃-Met) and added to the culture medium for metabolic labeling of cells, and D₃-m1A was subsequently detected in mRNA, indicating that SAM is the methyl donor for m1A methylation modifications.⁷ Most of the currently known methylated nucleotides in RNA (including m6A, m1A, and m7G) are synthesized and installed by various methyltransferases using SAM as a universal methyl donor.^7,8

Table 1.

The m1A sites in different organisms as well as the associated MTases

RNA class	Species/genus	Nt-position/mRNA	m1A writer	References
mt-tRNA	Homo sapiens	9	TRMT10C	Vilardo et al.54
tRNA	Sulfolobus acidocaldarius	9	Saci_1677p	Kempenaers et al.55
tRNA	Thermococcus kodakarensis	9	TK0422p	Kempenaers et al.55
tRNA	Rattus norvegicus (Sprague-Dawley)	14	Unknown	Salas and Dirheimer56
tRNA	B. subtilis	22	TrmK	Roovers et al.57
cyt-tRNA	Yeast/H. sapiens	58	TRMT6/61A	Anderson et al.10; Ozanick et al.50
mt-tRNA	H. sapiens	58	TRMT61B	Chujo et al.58
tRNA	T. thermophilus P. abyssi	58	TrmI	Droogmans et al.59; Roovers et al.60
tRNA	Mycobacterium tuberculosis	58	Rv2118p	Varshney et al.61
28S rRNA	H. sapiens	1,322	NML (RRP8)	Waku et al.44
	Mus musculus (C57BL/6J)	1,136
	Schizosaccharomyces pombe	670		Sharma et al.46
25S rRNA	Candida albicans	643
	S. cerevisiae	645		Peifer et al.43
		2,142	BMT2	Sharma et al.28
16S mt-rRNA	vertebrates	947	TRMT61B	Bar-Yaacov et al.32
mt-mRNA	H. sapiens	mt-COX1/2/3 mt-CYB mt-ND4L	TRMT61B	Li et al.13
mt-mRNA	H. sapiens	mt-ND5 1374	TRMT10C	Li et al.13; Safra et al.30
cyt-mRNA	H. sapiens	a	TRMT6/61A	Li et al.13; Safra et al.30

nt, nucleotide; mt, mitochondria; cyt, cytoplasm.

^aTRMT6/61A-mediated m1A methylation is highly sequence specific and is responsible for the formation of m1A in mRNA by recognizing the GUUCRA motif, which is structurally similar to the tRNA TΨC loop.

Figure 3.

The molecular mechanism of m1A formation

In the nucleus, m1A methylation is installed by TRMT6/TRMT61A, NML, and BMT2. In mitochondria, m1A is installed by TRMT10C and TRMT61B. The m1A modification is removed by the demethylases FTO, ALKBH1, and ALKBH3. The reader protein recognizes m1A sites and determines the fate of the target RNA.

Site 645 of S. cerevisiae 25S rRNA corresponds to site 1,322 of human 28S rRNA and site 1,136 of mouse 28S rRNA, and NML can add m1A at the aforementioned three sites of rRNA.^43,44 In contrast, BMT2 is responsible for catalyzing m1A formation at site 2,142 in S. cerevisiae 25S rRNA, site 3,625 in human 28S rRNA, and site 3,301 in mouse 28S rRNA.^28,44 In human mitochondria, TRMT61B catalyzes the formation of 16S mt-rRNA m1A947. Thus, TRMT61B is the first methyltransferase that has been proved to catalyze the formation of m1A from both tRNAs and rRNAs (Table 1, Figure 3).³²

TRMT6 and TRMT61A associate with each other to form a methyltransferase complex, and, individually, they show different subcellular localizations, with TRMT61A distributed in both the cytoplasm and nucleus, and TRMT6 strictly localized in the nucleus. TRMT61A is transferred to the nucleus and binds to TRMT6 to exert methyltransferase activity, with TRMT61A being responsible for binding SAM and completing catalytic function, while TRMT6 is critical for tRNA binding; furthermore, both cooperate to participate in the formation of the m1A58 modification in tRNA (Table 1; Figure 3).^10,50,62 In human mitochondria, TRMT10C generates m1A as well as m1G at position 9 in mt-tRNAs, whereas TRMT61B is responsible for m1A methylation at position 58 in some mt-tRNAs (Table 1; Figure 3).^30,54,58 In Thermus thermophilus HB27 (bacteria) and Pyrococcus abyssi (Archaea), TrmI—a homolog of TRMT61A—catalyzes the formation of tRNA m1A58 without other auxiliary proteins.^59,60 The m1A22 MTase (i.e., TrmK) found in B. subtilis belongs to the COG2384 protein family and is encoded by the YqfN gene. TrmK is conserved in gram-positive bacteria and is observed in some gram-negative bacteria; however, no TrmK homologs have been identified in eukaryotes to date. TrmK is well conserved in the bacterial kingdom and shows a high sequence identity (>40%) in several pathogenic bacteria (e.g., Staphylococcus aureus, Vibrio cholerae, Streptococcus pneumoniae, Listeria monocytogenes).⁵⁷ The activity of m1A14 Mtase has not yet been determined; however, m1A14 MTase activity was identified in extracts prepared from rat brain cortices and the enzyme was partially purified; however, the gene encoding the protein remains unknown.⁵⁶

With the continuous development of m1A sequencing technology, the m1A sites on mRNA have been gradually recognized. Li et al.¹³ showed that TRMT61B specifically catalyzes the production of m1A modifications in some mt-mRNAs, including mt-COX1, mt-COX2, mt-COX3, mt-CYB, and mt-ND4L (Table 1, Figure 3). TRMT6/TRMT61A-mediated m1A methylation is highly sequence specific and forms m1A in mRNA by recognizing the GUUCRA (R denotes A or G) motif, which is structurally similar to the tRNA TΨC loop (Table 1, Figure 3).^13,30 TRMT10C catalyzes the formation of mt-ND5 m1A1374, with m1A modification levels that are extremely tissue specific and tightly developmentally controlled.³⁰ No mRNA-specific m1A MTase has been identified to date.

In 2020, Scheitl et al.⁸ screened the world’s first non-protein methyltransferase, the ribozyme, using systematic evolution of ligands by exponential enrichment (SELEX) technology and named it MTR1. The study was groundbreaking because they discovered that the ribozyme MTR1 can target RNA by base complementary pairing and use free m6G as a methyl donor to achieve m1A methylation modification at specific sites of the target RNA.

Erasers

The first reversible RNA modification identified was m6A. m6A can be demethylated by two AlkB family proteins: FTO⁶³ (also known as ALKBH9) and ALKBH5.⁶⁴ Similarly, three AlkB family members, ALKBH1,^9,65 ALKBH3,^29,66,67 and ALKBH7,⁶⁸ and FTO⁵¹ have been identified so far to demethylate m1A in RNA (Figure 3).

In 2016, ALKBH1 was demonstrated to remove the m1A modifications from tRNA in human cells.⁹ To analyze the observed ALKBH1 demethylation, ALKBH1 was transiently overexpressed or knocked down in HeLa cells, and the levels of m1A were quantified using liquid chromatography-tandem mass spectrometry (LC-MS/MS). Transient knockdown of ALKBH1 increased the m1A levels in total tRNA by ∼6% compared with the corresponding controls, whereas m1A levels in rRNA were not affected. In the overexpressed samples, an ∼16% decrease in the m1A methylation levels in total tRNA was observed, whereas the m1A methylation levels in mRNA only slightly decreased. The above results suggest that ALKBH1 may also mediate the demethylation of m1A in mRNA, but not as specifically as it does for tRNA m1A. Further examination of all tRNAs enriched in the crosslinking and immunoprecipitation (CLIP) product of ALKBH1 observed that all of them contain the m1A58 modification, suggesting that m1A58 in tRNAs is the main substrate for ALKBH1. Subsequent protein sequence alignment of ALKBH1 with known tRNA-binding motifs revealed that ALKBH1 has a tRNA-binding domain, and, considering that the substrate m1A58 is located within the tRNA TΨC loop, it was believed that ALKBH1 is selective for the TΨC loop and preferentially mediates m1A58 demethylation in tRNA.

ALKBH1 is similarly involved in the demethylation of the mitochondrial tRNA m1A58.⁶⁵ In addition, ALKBH1 is involved in the biosynthesis of the f5C modification at position 34 (f5C34, the first position of the anticodon, also called the wobble position, Figure 1B) in cytoplasmic tRNA^Leu and mitochondrial tRNA^Met. The presence of f5C34 stabilizes the non-Watson-Crick pairing of the first position of the anticodon with the third position of the codon, and the f5C34 modification of mt-tRNA^Met is essential for the translation of AUA, a non-universal codon in mammalian mitochondria. The universal codon AUA encodes isoleucine, whereas the mammalian mitochondrial codon AUA encodes methionine.⁶⁹ In mammals, mitochondria only use one tRNA (tRNA^Met_CAU) to read the AUG and AUA codons as methionine, so the presence of f5C34 enables mt-tRNA^Met (with the anticodon f5CAU) to recognize codons AUA and AUG.^70,71,72 After knockdown of ALKBH1, HEK293T cells exhibited a strong reduction in mitochondrial translation and reduced respiratory complex activity, suggesting that ALKBH1 has additional tRNA-modifying functions in different cellular compartments, and its mediated f5C formation is required for normal mitochondrial function.^65,73

Certain exogenous chemical reagents and endogenous metabolites can disrupt Watson-Crick base pairing by continuously producing various alkylated base modifications (such as m1A, m3C, and m1G), thereby destroying the spatial structure of DNA, causing DNA damage, leading to gene mutation, inducing cytotoxicity, and causing cell death.^74,75,76 Previous studies have shown that ALKBH3 is mainly responsible for catalyzing the demethylation of alkylation modifications, such as m1A and m3C, on ssDNA or RNA in humans, thereby protecting the cellular genome from alkylation damage and playing a crucial role in maintaining the normal structure of DNA and realizing the normal physiological function of cells.^77,78,79 Discovered in 2016, ALKBH3 could reduce the m1A modification of mRNA in HEK293T cells, and its specifically recognized m1A sites were mainly distributed in the 5′ UTR of mRNA, indicating that ALKBH3 has transcriptional regulatory functions in addition to RNA alkylation repair functions.²⁹ ALKBH3 is currently the only known m1A eraser in mRNA. In HeLa cells, tRNA was found to be the main RNA type bound by ALKBH3. The protein sequence alignment of ALKBH3 with the tRNA-binding domain of ALKBH1 showed that ALBKH3 contains a similar tRNA-binding domain, which may function in tRNA recognition.⁶⁷ Taken together, the above studies indicate that ALKBH3 can remove the m1A modification on both mRNA and tRNA.

FTO was the first RNA demethylase to be identified. In 2011, it was first reported that it could erase m6A modifications inside mRNAs,⁶³ thereby pioneering and triggering a research boom in epitranscriptomics. Recent research has led to a new understanding of FTO and revealed that FTO cannot only reversibly remove the methylation modification of m6A but can also significantly reduce the levels of m1A58 modification in nuclear and cytoplasmic tRNA, thereby effectively inhibiting protein translation.⁵¹

In 2021, Chuan He’s team⁶⁸ discovered that ALKBH7 can highly specifically erase m₂²G26 on Ile-tRNA and m1A58 on Leu1-tRNA in mitochondria, whereas it does not have any effect on the known m1A, m1G (N1-methylguanosine), and m₂²G (N2,N2-dimethylguanosine) modifications on other mt-tRNA, mt-rRNA, and mt-mRNA.

Readers

The dynamic and reversible regulation of the m1A modification is determined by the functional interaction between m1A writers and erasers. However, m1A must be recognized by various readers for different downstream biological functions. Compared with the research on m1A writers and erasers, related research on m1A readers is lagging. Previous studies have shown that the YTH domain protein family can regulate RNA signaling pathways, including RNA splicing,⁸⁰ RNA stability,⁸¹ protein translation,⁸² and nuclear export,⁸³ by recognizing the m6A modifications of target mRNAs and then directing different complexes. Currently, part of the YTH domain protein family, including YTHDF1, YTHDF2, YTHDF3, and YTHDC1,^47,48,49 has been discovered to also bind to various m1A-modified sequences in a methylation-specific manner (Figure 3). YTHDF2 recognizes a specific m1A site of target genes through its C-terminal YTH domain and transports the recruited RNA to the P body through its N-terminal domain, accelerating the degradation of m1A-modified RNA.^47,49 YTHDF1 promotes the translation of m1A-modified mRNAs in the cytoplasm.⁴⁹ It is currently considered that YTHDF3 plays a synergistic role, cooperating with YTHDF1 to promote translation or accelerating the degradation of m1A-modified transcripts through direct interaction with YTHDF2.⁴⁸ Compared with YTHDF1/2, YTHDC1 binds rather less tightly to m1A-modified mRNAs,⁴⁷ and more evidence is required to support the recognition of m1A modification by YTHDC1 in the future.

Biological functions of m1A

Unlike m6A, methylation at the N1 position of adenosine occurs in the Watson-Crick edge and produces a positive charge.⁸⁴ Although m1A modification is far less abundant than m6A modification in human and mouse tissues, given that this modification introduces a positive charge, it is shown that it can significantly alter the local or overall structure of RNA and protein-RNA interactions.⁸⁵ For instance, the electrochemical interaction generated by the positive charge of m1A is essential for the structure and function of tRNA.⁴² The m1A modification in mRNA blocks Watson-Crick base pairing, thereby affecting reverse transcription and protein translation.^86,87 In addition, the distribution of m6A and m1A on RNA differs. m6A is concentrated in the 3′ UTRs and around the stop codon (400-nucleotide window centered on the stop codon),⁸⁸ whereas m1A is enriched mostly in the 5′ UTRs and dramatically around the start codon (50-nucleotide window centered on the start codon).²⁹ Because the spatial structure, charge, and distribution of these two alterations differ (Figures 4A–4D), m1A has different biological activities than m6A.

Figure 4.

Comparison of m1A and m6A

(A) The distribution of m1A and m6A on mRNA. The dimensions of the circles are a schematic representation of the amount of m1A or m6A modification present in the transcript (for example, m6A is mainly enriched close to the stop codon area, m1A is sharply positioned around the start codon, m7G is highly abundant at the cap, and m6Am is located exclusively in the 5′ cap of eukaryotic mRNAs). (B) Consensus motifs for m1A and m6A. (C) Writers, erasers, and readers of m1A and m6A. NML and BMT2 are rRNA-specific m1A Mtases, and ZCCHC4 and METTL5 are rRNA-specific m6A Mtases. (D) Biological function of m1A and m6A.

m1A maintains the stability of tRNA and a high heat resistance

The m1A58 modification of tRNAs is considerably conserved in bacteria, Archaea, and eukaryotes, underscoring the significance of the m1A58 modification. In S. cerevisiae, m1A58 methylation in tRNA_i^Met is essential for maintaining the stability of its tertiary structure (inverted L-shape structure), and loss of m1A methylation results in polyadenylation by the Trf4/Air2/Mtr4 polyadenylation (TRAMP) complex (the complex containing the poly(A) polymerase Trf4p, the Zn-knuckle protein Air2p, and the RNA helicase Mtr4p) and subsequent degradation of tRNA_i^Met by the exonuclease Rex1p and exosomes.^9,10,89 The m1A58 methylation in human tRNA₃^Lys is particularly important for HIV replication, and HIV-1 replication in vivo is inhibited when the adenosine residue at position 58 is replaced with a uridine nucleoside.^90,91

The structural thermostability of tRNAs in thermophilic bacteria is closely associated with post-transcriptional modifications. The combined effect of m1A58, Gm18 (2′-O-methylguanosine at position 18), s2T54 (2-thioribothymidine at position 54), and m7G48 (7-methylguanosine at position 48) modifications in tRNAs was shown to increase the melting temperature of tRNAs from thermophilic bacteria by ∼9°C compared with that of unmodified tRNAs.^{59,92,93,94,95} TrmI—the methyltransferase of m1A58—was found to be unable to grow at 80°C when it was inhibited, indicating that the m1A58 modification in tRNA has an important role in stabilizing tRNA structure and ensuring bacterial growth under high-temperature conditions.⁵⁹ Moreover, in Archaea, the m1A9 Mtase is only present in hyperthermophiles and is absent in mesophilic archaeons, indicating that m1A9 may also contribute to maintaining the thermostability of archaeal tRNAs.⁵⁵

In addition to stabilizing the tertiary structure of tRNA, m1A promotes the correct folding of the tRNA secondary structure (clover) for its normal biological function. The m1A9 modification of human mitochondrial tRNA^Lys is essential for correct folding of tRNA^Lys, and the lack of m1A9 prevents tRNA^Lys from folding into the standard cloverleaf structure.⁴¹

Until now, little was known regarding the specific roles of m1A14 and m1A22 in tRNA, but the tRNA m1A22 Mtase is critical for bacterial survival in S. pneumoniae.⁹⁶

m1A is involved in the protein translation process

In 2017, single-nucleotide-resolution m1A sequencing technology was developed for studying the localization and function of m1A in human whole transcription.¹³ The study showed that 58.6% of m1A modifications were located in the 5′ UTR of the mRNA, which in turn contained 8% of m1A occurring on the first nucleotide (cap + 1) at the 5′ end. Ribosomal analysis revealed that m1A modification inside the 5′ UTR promoted translation by enhancing mRNA stability, and this promotion was even higher for m1A at the cap + 1 position, whereas no effect on translation was observed for m1A modification located in the CDS or 3′ UTR.¹³

Unlike nuclear-encoded mRNAs, m1A modifications in mt-mRNA are almost exclusively located in the CDS region, whereas the translation of proteins in mitochondria depends on base pairing between the codon of mt-mRNA and the anticodon of the cognate mt-tRNA, especially the complementary pairing of the first and second nucleotide bases in the codon with the third and second nucleotide bases in the anticodon, respectively. The positive charge introduced by the m1A modification blocks Watson-Crick A-U pairing.⁸⁶ Based on the above studies, overexpression of TRMT61B led to a significant increase in the m1A level of the target gene mRNA while decreasing the protein translation level, thereby indicating that the m1A modification in mt-mRNA greatly interferes with protein translation in mitochondria.¹³ You et al.⁹⁷ verified in vitro translation by synthetic mRNA sequences and indicated that the m1A modification at the first or second position of the codon in the CDS region of cytoplasmic mRNA significantly reduced the translation efficiency of proteins in both E. coli and wheat germ using mass spectrometry (MS), whereas the m1A modification at the third position of codons had a reduced inhibition efficiency on translation. This observation hints that the m1A modification may have different biological functions within different regions of different mRNAs. When m1A is located in the 5′ UTR of cytoplasmic mRNAs, it is considered to promote translation by enhancing mRNA stability. In contrast, when it is located in the CDS of the mt-mRNA, m1A prevents the base complementary pairing of codons and anticodons, inhibiting the protein translation process.

In addition to m1A modification in mRNA, m1A modifications in tRNA and rRNA are similarly involved in the translation process. ALKBH1 can affect the stability of tRNA_i^Met by mediating the demethylation of m1A58, thereby significantly promoting translation initiation and cell proliferation. Transient knockdown of ALKBH1 in HeLa cells significantly increased the level of m1A modifications in tRNAs, and m1A-modified tRNAs were preferentially recognized and recruited to the active translation complexes, promoting translation elongation and protein synthesis.⁹

Modifications of m1A645 on cytoplasmic 25S rRNA and m1A947 on mitochondrial 16S rRNA play a role in stabilizing the rRNA structure, which is required for the formation of the translation initiation complex.^32,46 Erasing the m1A947 modification on 16S mt-rRNA revealed inefficient protein translation and slow cell growth, indicating that the m1A modification at this site is particularly critical for protein translation and cell development and growth.³² In mitochondria, TRMT61B simultaneously acts as an mt-tRNA, mt-rRNA, and mt-mRNA MTase, forming m1A modifications at specific sites. The m1A modification in mt-tRNA and mt-rRNA can promote translation by stabilizing their respective structures, but m1A modifications in mt-mRNA affect binding to mt-tRNA and inhibit translation. The changes in protein synthesis levels upon TRMT61B knockdown are likely the result of a mixed effect of m1A modifications in these translation components (rRNA, tRNA, and mRNA). Because both m1A947 in 16S mt-rRNA and m1A58 in mt-tRNA are highly modified sites, when TRMT61B is overexpressed, m1A levels in mRNA more significantly increased than in these ncRNAs, and the corresponding levels of proteins encoded by target mRNA were reduced, while the levels of proteins encoded by non-target mRNA were hardly affected. In contrast, after TRMT61B knockdown, the levels of proteins encoded by non-target mRNAs were decreased, probably due to a decrease in the level of m1A modification in mt-tRNA and mt-rRNA, resulting in structural instability and inhibition of translation.³²

Mitochondrial RNA (mt-RNA) has a unique transcription, processing, and maturation process.^98,99,100 Rapid bidirectional transcription on mitochondrial DNA (mtDNA) produces two long-stranded complementary unprocessed polycistronic RNAs, resulting in mitochondrial double-stranded RNA (dsRNA).^98,99,100 In this series of immature mt-RNAs, 13 mt-mRNAs, 22 mt-tRNAs, and two mt-rRNAs are linked together,^98,99 and mt-tRNAs act as junction sites, spacing different mRNAs apart; all mt-mRNAs and mt-tRNAs will be cleaved and released from the long-stranded mitochondrial polycistronic RNAs during further processing and maturation.¹⁰¹ ALKBH7 mainly regulates the demethylation of the Ile-tRNA and Leu1-tRNA regions on mitochondrial polycistronic RNAs. By knocking down ALKBH7, researchers observed that elevated methylation levels of m₂²G26 on the Ile-tRNA and m1A58 on the Leu1-tRNA accelerated the cleavage rate at almost all tRNA junctions. The abnormally accelerated cleavage and processing of polycistronic RNAs resulted in the degradation of pre-tRNA and nascent mRNA, which ultimately led to a reduction in the content of most mature mt-tRNAs, thereby attenuating the mitochondrial translation process, reducing mitochondrial protein levels, and weakening mitochondrial respiratory metabolism.⁶⁸

Elongation factor thermo unstable (EF-Tu), a subunit of EF, is responsible for mediating the entry of aminoacyl-tRNA into the aminoacyl site (A site) of the ribosome, which in turn facilitates mRNA translation and polypeptide chain extension. In bacteria, the elongation factor acts mainly by binding to two regions of tRNA: the terminal region of the amino acid arm and the TψC loop.¹⁰² However, most mt-tRNAs in nematodes lack the TψC loop.^103,104,105 Follow-up studies have revealed that almost all mt-tRNAs in nematodes lacking the TψC loop had a high abundance of m1A9 modifications.^104,105 The m1A9 modification of mt-tRNA is necessary for the binding of EF-Tu to mt-tRNAs in nematodes that lack the TψC loop, possibly because m1A9 stabilizes the secondary structure of the tRNA⁴¹ or m1A9 can interact with the C-terminal amino acid residues of EF-Tu.¹⁰⁶

The development of m1A detecting methodology

The concentration of modified nucleosides in RNA molecules or human body fluids can be measured by at least 14 techniques, including ELISA, liquid chromatography (LC)-MS, gas chromatography (GC)-MS, electrospray ionization (ESI)-MS, ultra-high performance liquid chromatography-quadrupole time-of-flight (UHPLC-Q-TOF)-MS, high-pressure liquid chromatography (HPLC), reverse-phase (RP)-HPLC, HPLC-MS, and capillary electrophoresis (CE).^{28,107,108,109,110,111}

Earlier, ELISA was used to quantify m1A content in the urine of tumor patients.^112,113 In recent years, LC-MS and HPLC-MS have been combined to quantify m1A modifications in yeast RNA.^9,28 RP-HPLC allows the easy and rapid separation of typical nucleosides (A, U, G, and C) as well as other modified nucleosides.¹¹⁴ Furthermore, only a small amount of sample is required for detection without expensive MS detectors.^114,115 However, RP-HPLC consumes large amounts of solvent.¹¹⁶ LC-MS or HPLC-MS can sensitively analyze trace amounts of modified nucleosides on RNA from different sources¹¹⁷ and complete the determination of modified nucleosides in a few micrograms of RNA in a short time.¹¹⁸ However, LC-MS and HPLC-MS are complicated and not sensitive enough to accurately detect m1A modifications in mRNA.

Considering the very low abundance of m1A in mRNA, to investigate the m1A methylation of the transcriptome in detail, extremely sensitive m1A high-throughput sequencing must be developed. Furthermore, under alkaline conditions, m1A can be rearranged to m6A (Dimroth rearrangement).¹¹⁹ Thus, during the preparation of mRNA fragmentation or mRNA purification, the signature of m1A in mRNA may vanish, making it difficult to reduce the noise in m1A sequencing. The methyl group on m1A in mRNA effectively inhibits reverse transcription by blocking Watson-Crick base pairing, resulting in truncations or mutations in reverse transcription products.¹²⁰ Because this feature can be utilized to detect m1A sites, high-throughput m1A sequencing was created with this aim in mind. Two independent teams have detected and analyzed m1A sites on mRNAs using first-generation m1A sequencing technology and mapped the m1A RNA methylomes with a ∼130-nucleotide resolution (Figures 5A and 5B).^7,29 Dominissini et al.⁷ analyzed human and mouse purified mRNA and revealed that the m1A/A ratios ranged from approximately 0.015% to 0.054% in cell lines, such as HEK293, HepG2, HeLa, mouse embryonic fibroblasts (mEFs), and mouse embryonic stem cells (mESCs), whereas the ratio was higher in tissues such as mouse brain and kidney, reaching 0.16%. In the transcription of HEK293T cells, Li et al.²⁹ identified 901 high-confidence m1A peaks, including 841 and 46 peaks found in mRNA and lncRNA transcripts, respectively. These m1A peaks originate from 887 transcripts, which are encoded by 600 human genes. The bulk of these m1A sites in these mRNAs were significantly enriched within the 5′ UTR and near start codons.

Figure 5.

m1A mapping and measuring techniques

(A and B) Both m1A-ID-seq and m1A-MeRIP-seq use reverse transcription termination and antibody enrichment to detect m1A, but they treat negative controls differently. (A) The m1A-MeRIP-seq uses Dimroth rearrangement to change m1A to m6A, which has no effect on pairing and eliminates the impact of m1A on reverse transcription. (B) In contrast, the m1A-ID-seq uses demethylase to remove m1A. (C and D) Both m1A-seq and m1A-MAP detect m1A by using TGIRT. (C) The m1A-seq has a similar library building process to m1A-MeRIP-seq. (D) The m1A-MAP has a similar library building process to m1A-ID-seq. In m1A-MAP, a unique molecular identifier (UMI) at the 3′-adaptor is established, which eliminates duplication caused by PCR and improves the accuracy of detection. In m1A-MAP, HIV RT-1306 can also be used to replace TGIRT, which has a more powerful readthrough efficiency and a higher mutation frequency.

The sequencing methods of the above two teams are similar in that they both used m1A-specific antibodies to enrich m1A-modified RNA fragments. There are also slight differences between the two methods in the subsequent library building assays. Dominissini et al.⁷ used the Dimroth rearrangement method to convert m1A to m6A in the collected RNA fragments, followed by cDNA library sequencing along with the untreated RNA fragments. Because cDNA in the untreated group was mismatched or truncated, whereas the cDNA in the Dimroth-rearrangement-treated group was intact, comparing the sequencing results of the two groups resulted in high-confidence m1A peaks based on mismatch rates (Figure 5B). Li et al.²⁹ used ALKBH3 from E. coli to erase the m1A methylation in the RNA, followed by library building sequencing together with the RNA without ALKBH3 treatment (Figure 5A).

The abortion site during cDNA synthesis does not accurately reflect the m1A modification site and requires a reverse transcriptase (RT) with greater synthetic power to read through the m1A-containing fragment and introduce mutations at the m1A site. Thus, the researchers created the second-generation m1A sequencing technique to more accurately identify m1A sites for a more accurate picture of m1A distribution. To optimize the reverse transcription step, a high-performance RT termed thermostable group II intron RT (TGIRT) is used in the second-generation m1A sequencing approach.^13,30 Compared with other RTs, TGIRT has a longer RT domain and thumb domain, and numerous studies have confirmed that the longer RT domain and thumb domain enable more extensive interaction with the RNA template, resulting in more excellent readthrough during reverse transcription.^121,122 Furthermore, it was discovered that TGIRT has an even higher mismatch rate of 94% when recognizing the m1A site.¹²³ TGIRT can read through RNA fragments containing m1A and can introduce mutations at the m1A site after testing, demonstrating excellent readthrough efficiency and a relatively high mutation frequency at the m1A site. This results in a stronger signal-to-noise ratio for the precise position of m1A and subsequently obtains single-base resolution (Figures 5C and 5D).

Using second-generation m1A sequencing methods, Li et al.¹³ identified 418 m1A sites in the mRNA of HEK293T cells (Figure 5D). The m1A modification was significantly enriched in the 5′ UTR, followed by that in the CDS region, with a tiny amount located in the 3′ UTR, which is consistent with previous findings.^7,29 Surprisingly, around the same time, another team took a similar approach but came to very different conclusions (Figure 5C). Safra et al.³⁰ identified only 14 m1A sites in mRNAs, with nine in cytoplasmic mRNAs and five in mt-mRNAs, and they concluded that m1A is a rare modification in human mRNAs. The reason for this discrepancy may be that (1) the alkaline conditions used for Dimroth rearrangement may affect RNA integrity compared with ALKBH3 demethylation; (2) the unique molecular identifier (UMI) used by Li et al. at the 3′ adaptor can eliminate duplication caused by PCR and improve the accuracy of detection; and (3) TGIRT does not have sufficient readthrough activity for m1A-containing RNA fragments and detecting m1A in tRNA, and rRNA may have little effect (high m1A content). However, the m1A content in mRNA is low, which is prone to inconsistent results due to insufficient experimental coverage. To more accurately and comprehensively detect m1A modifications in mRNA, in 2019, Zhou et al.¹²⁴ engineered the HIV-derived RT such that the modified RT (HIV RT-1306) has a stronger readthrough ability for m1A modifications, can cross m1A to produce intact cDNA products, and can insert precisely mutated bases at the m1A site. This mutation information can be used to pinpoint the location of m1A in the transcriptome in second-generation sequencing technology and to estimate the amount of m1A in this mRNA, ultimately identifying a large number of m1A sites that may be present in human mRNA but may have low stoichiometry. Finally, in the interest of space, we summarize the fundamentals, advantages, and limitations of the above m1A measuring techniques in Table 2.

Table 2.

m1A mapping and measuring techniques

Techniques	Fundamentals	Advantages	Limitations
LC-MS	digestion to single nucleotide and UV detection of m1A based on its physicochemical properties	•quantitative method •standardized method •short test time	•no sequence context and localization information •inability to distinguish the source of m1A •not high enough sensitivity
m1A-ID-seq	immunoprecipitation of m1A-enriched fragments using anti-m1A antibody; the enriched fragments were subjected to demethylase or mock treatment. Identification of the m1A sites within the peaks by using a truncation signature	•compared with Dimroth rearrangement, AlkB ensures RNA integrity as much as possible •low number of laborious steps	•inability to call m1A sites with single-nucleotide resolution •inability to call multiple m1A sites within peaks •E. coli AlkB does not erase all m1A sites
m1A-MeRIP-seq	immunoprecipitation of m1A-enriched fragments using anti-m1A antibody. m1A causes both reverse transcription stops and mismatch generation, and m1A was converted to m6A by using the Dimroth rearrangement method, followed by analysis of the m1A sites according to the mismatch rate	•low number of laborious steps •improved resolution for m1A site recognition	•the alkaline conditions used for Dimroth rearrangement may affect RNA integrity •inability to call m1A sites with single-nucleotide resolution •Inability to call multiple m1A sites within peaks
m1A-seq	immunoprecipitation of m1A-enriched fragments using anti-m1A antibody, TGIRT induced by RT at m1A sites, the remaining library building process is the same as m1A-MeRIP-seq	•TGIRT has higher processivity and mutation frequency at m1A sites •achieving single-base resolution •ability to call multiple m1A sites on RNA	•the alkaline conditions used for Dimroth rearrangement may affect RNA integrity •the lack of UMIs could lead to trouble in data analysis and mutation rate calculation
m1A-MAP	immunoprecipitation of m1A-enriched fragments using anti-m1A antibody, TGIRT/HIV RT-1306 induced by RT at m1A sites; the remaining library building process is the same as m1A-ID-seq	•achieved single-base resolution •the UMI used at the 3′-adaptor can eliminate repetition caused by PCR and improve the accuracy of detection •HIV RT-1306 has more powerful readthrough efficiency and higher mutation frequency	•TGIRT does not have sufficient readthrough activity for m1A-containing RNA fragments

Role of m1A in development and various pathogenesis

m1A is involved in the regulation of growth and development

Numerous studies have shown that m1A plays an important role in embryonic growth and development (Figure 6). In eukaryotic cells, ribosome biosynthesis requires large amounts of energy, and it can undergo adaptive regulation in response to changes in the intracellular energy status. When cells are under low-glucose conditions, NML can regulate intracellular energy metabolism by inhibiting rDNA transcription and ribosome biosynthesis through its N-terminal region to reduce intracellular ATP consumption.¹²⁵ Follow-up studies revealed that NML can also act as a 28S rRNA MTase through its C-terminal region, using SAM to regulate m1A modification in 28S rRNA, and, when NML is depleted intracellularly, ribosomal 60S subunit formation is impaired and later degraded via the ubiquitin-proteasome pathway.^44,126 Furthermore, researchers observed that NML depletion enhanced RPL11-MDM2 interaction and increased p53 protein levels, thereby activating the p53 pathway and inducing cell death.⁴⁴ Taken together, these findings suggest that NML functions as an m1A methyltransferase of rRNA under normal conditions and reduces ATP consumption by inhibiting rDNA transcription under glucose-deprivation conditions. Thus, NML adapts to environmental changes by regulating the different steps of ribosome biogenesis, including transcription of rDNA as well as m1A methylation of rRNA.

Figure 6.

The potential role of m1A in growth and development

∗NML depletion enhances RPL11-MDM2 interaction, which activates the p53 pathway and induces cell death, and it remains to be verified whether it acts by regulating m1A in this process.

NML is essential for the survival of mice, and only 10%–20% NML-knockout mice can grow to adulthood. In surviving NML-knockout adult mice, because the repression effect of NML on rDNA transcription is relieved, high-fat-diet (HFD)-induced rRNA levels and AMP/ATP ratios in the liver were significantly increased. Here, increased rRNA transcription promoted protein synthesis and further accelerated energy expenditure, so higher energy expenditure could lead to resistance to HFD-induced obesity.¹²⁷ Compared with wild-type (WT) mice, NML-knockout mice were leaner in weight. As mentioned earlier, NML-knockout resulted in decreased m1A levels in 28S rRNA, which induced dysfunction in the 60S ribosome subunit, leading to increased protein degradation. Protein degradation is also the main intracellular ATP consumption pathway, which provides more evidence that NML-knockout mice can resist HFD-induced obesity.¹²⁷ Most remaining NML-knockout mice die before or shortly after birth, and the main cause of death is the discovery of impaired embryonic erythropoiesis with severe hematopoietic dysfunction. In NML-knockout embryos, rRNA levels were unaffected, but the m1A level in 28S rRNA was significantly reduced. Impaired ribosome biogenesis results in severely impaired erythropoiesis during embryonic development.¹⁴

In Drosophila, knockdown of TRMT10C, which mediates the formation of mt-tRNA m1A9 modification, results in embryonic lethality.¹²⁸ In HeLa cells, knockdown of TRMT10C leads to reduced mitochondrial respiration, revealing that it is very important for cell viability.¹²⁹ The very rare autosomal recessive disorder with oxidative phosphorylation deficiency 30 (COXPD30, MIM#616974) has recently been attributed to missense mutations of human mitochondrial TRMT10C. The patients presented with hypotonia, lactic acidosis, feeding difficulties, and deafness at birth and died of respiratory failure at about 5 months of age. However, in this study, considering that TRMT10C is not only responsible for m1A9 modifications in mt-tRNA but also participates in the processing of the 5′ end of mt-tRNA as a subunit of mitochondrial ribonuclease, it is difficult to attribute the patient’s phenotype solely to the m1A9 Mtase activity of TRMT10C.¹³⁰

TRMT6/TRMT61A is involved in embryonic development of Arabidopsis in addition to being responsible for m1A58 modification in tRNA and maintaining tRNA_i^Met stability.^15,131 Deficiency in either of the complex subunits TRMT6/TRMT61A would result in early embryo and endosperm developmental arrest, indicating that the complex plays a critical role in embryo development. Plant embryos require large and rapid protein synthesis during development and therefore require a large supply of tRNA. Embryonic lethality due to TRMT6/TRMT61A knockout may be caused by the lack of tRNA_i^Met.¹⁵ Plant seed development is tightly regulated, and a recent study showed that plants can precisely regulate the expression of TRMT61A through the MEKK1-MKK1/2-MPK4 cascade signaling pathway, which regulates Arabidopsis embryo and endosperm development.¹³¹ Unlike mammals, in Petunia, m1A modifications are most abundant in mRNA and are mainly enriched in the CDS region. m1A modifications in mRNA are closely associated with senescence in Petunia flowers and development in leaves.¹³²

During pregnancy, the maternal-fetal interface cells are exposed to various external environmental stimuli (such as hypoxia, lipopolysaccharides, and hormones), and the level of cellular metabolism is dynamically altered; in particular, a hypoxic environment significantly affects the activity of trophoblast cells.¹³³ Under hypoxic conditions, YTHDF3 is significantly downregulated in trophoblast cells. YTHDF3, a reader of m1A, promotes degradation of IGF1R mRNA by recognizing and binding to the m1A modification site on insulin-like growth factor 1 receptor (IGF1R) mRNA, inhibiting the activation of its downstream matrix metallopeptidase 9/2 (MMP9/MMP2) signaling pathway, thereby reducing the migration, invasion, and proliferation of trophoblast cells.⁴⁸ Thus, the downregulation of YTHDF3 caused by hypoxia significantly promotes migration and invasion of trophoblast cells.

m1A is involved in the regulation to stress responses

Under physiological conditions, the level of m1A modification in mRNA is not static. Numerous studies have revealed that the level of m1A modification can be dynamically regulated under different stress conditions. Dominissini et al.⁷ treated HepG2 cells with glucose or amino acid starvation for 4 h and found that m1A levels in mRNA could be reduced 2- to 3-fold, whereas heat shock treatment for 4 h increased m1A levels by 1.5-fold. Furthermore, significant differences in m1A levels were observed in mRNA of different tissues of mice, with the highest m1A levels in the kidney and brain; WT mouse brain tissues had higher m1A levels, up to more than 75% than those in obese mouse brain tissues. In the case of serum starvation or H₂O₂ treatment, more inducible m1A peaks were identified in mRNA, and most peaks were stress specific.²⁹ In mitochondria, m1A1374 levels in mt-ND5 were upregulated under heat-shock or hypoxic conditions and significantly downregulated upon serum starvation or actinomycin intervention.¹³ The lack of m1A645 modification in cytoplasmic 25S rRNA sensitizes cells to paromomycin and affects the assembly of ribosome subunits, whereas m1A2142 modification increases cell sensitivity to hydrogen peroxide and anisomycin.^28,43 In all, these studies suggest that the level of internal m1A modifications, whether in nuclear-encoded or mitochondrial-encoded RNA, is dynamically altered under certain stress conditions or physiological states, and that different tissues can also adjust m1A levels to adapt to their own growth needs.

It has been long speculated that modified nucleosides on tRNAs may act as “biosensors” for external environmental and physiological changes to rapidly regulate gene expression at the translational level by sensing environmental changes.¹³⁴ Consistent with these speculations, the abundance of modified nucleosides in tRNA does change in response to various stress stimuli.¹³⁵ In nature, plants are particularly vulnerable to external environmental changes, such as drought, cold, and high salt stress. The levels of m1A modifications in rice and Arabidopsis tRNAs were significantly reduced under cold stress or salt stress. In addition, Am (2′-O-methyladenosine), Cm (2′-O-methylcytidine), and m7G in tRNA were also found to be involved in the response to environmental stress, along with m1A modification.¹⁶

tRNA halves (tiRNAs) are newly identified non-coding small RNAs produced by angiopoietin-specific cleaving of the mature tRNA anticodon loops when cells are subjected to stressful stimuli (e.g., oxidative stress, starvation or nutritional deficiency, hypoxia, and heat shock) (Figure 7A).¹³⁶ tiRNAs can play important roles in various biological processes by regulating mRNA stability¹³⁷ and protein translation^138,139 and acting as epigenetic regulators.¹⁴⁰ A study showed that ALKBH1 knockdown significantly attenuated the pro-apoptotic effects of sodium arsenite on rat neuroblastoma while reducing tiRNA levels in tumor cells.¹⁴¹ When eukaryotic cells are subjected to stress stimuli, mRNA translation is stalled and polysomes dissociate, producing large amounts of free mRNA. Stress granules (SGs) are dense, non-membrane structures formed from free mRNA in the cytoplasm and contain various translation initiation factors, RNA-binding proteins, and non-RNA-binding proteins. The formation of SGs is an adaptive regulatory mechanism of cells, which has antioxidant and anti-apoptotic effects.¹⁴² When the stress is relieved, SGs depolymerize, and the mRNA continues to participate in translation. A recent study showed that TRMT6/TRMT61A and m1A-enriched mRNA were significantly enriched in SGs, and, after TRMT6/TRMT61A depletion, impaired SG formation was observed, sensitizing HeLa cells to heat shock and arsenite stress stimuli.¹⁷ The above studies indicate that TRMT6/TRMT61A is involved in the formation of SGs and catalyzes the formation of m1A modifications in mRNA in SGs (Figure 7B). The m1A-modified mRNAs dissociate more efficiently from polysomes at the onset of stress, assemble more rapidly, and accelerate the translation process when stress is relieved.

Figure 7.

The potential role of m1A in stress response

(A) The biogenesis and classification of tsRNA. The ANG or Dicer enzymes cleave the T loop to generate tRF-3, and the ANG cleaves the anticodon loop to generate tiRNA. (B) TRMT6/TRMT61A is involved in the formation of SGs and catalyzes the formation of m1A modifications in mRNA in SGs. The m1A-modified mRNAs dissociate more efficiently from polysomes at the onset of stress, resulting in the rapid formation of SGs that protect mRNAs from degradation by P bodies.

Zebrafish are important model vertebrates in biomedical research and are widely used to study the development and progression of human diseases. Hypoxia-ischemia-induced brain injury is common in the perinatal period and is known as hypoxic-ischemic encephalopathy (HIE).¹⁴³ In addition, hypoxia is a key regulator of poor prognosis in both tumorigenesis and ischemic cerebral infarction. Zebrafish brain tissue mRNA contains abundant m1A, m5C, m6A, and m7G modifications, with m1A modifications being the most abundant. In hypoxia-treated zebrafish brain, the proportion of m1A decreases and responds to hypoxia induction by affecting the variable cleavage of RNA and regulating the expression of downstream related genes; however, how the downstream genes affect hypoxic cell activity and which underlying pathways and mechanisms mediate these responses are still far from being elucidated.¹⁴⁴

m1A is involved in tumorigenesis and metastasis

Numerous studies have highlighted that m1A modifications play an important role in various cancers, usually via writing or erasing m1A modifications in the mRNA of oncogenes or cancer suppressor genes and thereby regulating the expression of oncogenes or tumor suppressor genes by recognizing m1A by readers or preventing readers from recognizing m1A markers (Figure 8).

Figure 8.

The potential roles of m1A in cancer progression

The potential effect of m1A in cancer progression is reflected in the regulation of tumor-related gene expression. ∗ALKBH3 upregulation accelerates the G1/S phase transition and promotes tumor cell growth, but it remains to be verified whether the tumor-promoting effect is mediated by the regulation of m1A modifications. m1A methylation plays a critical role in the biogenesis of various RNAs. In this review, Xiong et al. systematically and comprehensively summarize the installation, removal, and recognition of m1A and emphasize the importance of m1A in both the growth of organisms and pathogenesis of various diseases, particularly cancers.

Numerous studies have found that ALKBH3 has important clinical significance in the diagnosis and treatment of various cancers. Compared with normal tissues, ALKBH3 is highly expressed in pancreatic cancer,¹⁴⁵ lung cancer,¹⁴⁶ urothelial carcinoma,¹⁴⁷ and renal cell carcinoma,¹⁴⁸ and has been identified as a marker for high-grade prostate cancer,^149,150 suggesting that ALKBH3 plays an important role in tumorigenesis and contributes to early diagnosis. In different cancer subtypes, ALKBH3 functions as an m1A demethylase and plays a tumorigenic role in cancer progression through multiple regulatory mechanisms. In breast and ovarian cancers, demethylation of m1A by ALKBH3 increased protein levels of CSF-1 by increasing the half-life of CSF-1 mRNA, and high levels of CSF-1 considerably increased the invasiveness of cancer cells.¹⁵¹ This study reported, for the first time, that m1A is also involved in determining mRNA stability. In lung cancer cells, as a tRNA m1A-modified demethylase, ALKBH3-mediated demethylation in tRNA is more sensitive to angiopoietin-dependent cleavage, followed by the generation of tDRs (tsRNAs) around the anticodon loop, which are involved in ALKBH3-mediated cancer progression by regulating ribosome assembly and increasing the translation rate to prevent cytochrome c (Cyt c)-triggered apoptotic pathways.⁶⁷ In addition, ALKBH3 can also improve the protein translation efficiency and promote protein synthesis in cancer cells by erasing m1A, m3C, and m6A methylation modifications in tRNAs.¹⁵²

Numerous molecules associated with ALKBH3 are involved in cancer metastasis or resistance to anticancer drugs. In human lung cancer, transient knockdown of ALKBH3 by siRNA transfection revealed that p21 and p27 were upregulated and tumor cell growth was strongly inhibited. Therefore, ALKBH3 knockdown may inhibit proliferation of human lung cancer cells through p21/p27-mediated cell-cycle arrest in the G1 phase.¹⁴⁶ As mentioned above, significant ALKBH3 upregulation has been observed in various cancers, and it is hypothesized that ALKBH3 upregulation accelerates the G1/S phase transition and promotes tumor cell growth; however, the exact molecular mechanism remains unknown. Furthermore, whether the tumor-promoting effect is mediated by the regulation of m1A modifications remains to be elucidated. ALKBH3 is also an important repair molecule for DNA alkylation damage^77,78,79; thus, it is hypothesized that ALKBH3 upregulation not only leads to massive proliferation of cancer cells during tumor formation but also maintains genomic integrity. It is well known that cancer cells stalled in the G1 phase are more sensitive to DNA damage reagents. Thus, ALKBH3 may also be one of the key molecules in determining the efficacy of cancer chemotherapy.

In addition to the identification of a large amount of ALKBH3 expression in cancer cells, in recent years, some studies have also found highly methylated modifications of CpG islands in the ALKBH3 promoter region in certain cancers (such as Hodgkin’s lymphoma¹⁵³ and breast cancer¹⁵⁴). The hypermethylation of CpG islands will lead to transcriptional silencing of its downstream genes, and, eventually, the ALKBH3 mRNA and its protein levels will be significantly reduced. In Hodgkin’s lymphoma, malignant Hodgkin cells and Reed-Sternberg cells (R-S cells) are surrounded by a large tumor microenvironment that plays a key role in tumors. Collagen is an important component of this microenvironment, and numerous studies have confirmed that increased collagen content significantly promotes the occurrence and progression of various cancers (such as pancreatic cancer,¹⁵⁵ breast cancer,¹⁵⁶ and lung cancer¹⁵⁷) Silencing of the ALKBH3 gene in Hodgkin’s lymphoma increases the m1A levels on Col1a2 and Col1a1 mRNAs, which promotes translational efficiency and greatly increases Col1a2 and Col1a1 protein levels. The increased collagen promotes the migration and invasion of Hodgkin’s lymphoma and reduces the overall survival of patients.¹⁵³

Shi et al.¹⁵⁸ analyzed urine samples from 32 patients with bladder cancer and 16 healthy volunteers and found that the m1A levels in the urine were significantly higher in patients with bladder cancer. The expression levels of TRMT6/TRMT61A in cancer tissues and adjacent tissues of the same patients were also detected by western blotting; the results showed that the expression of TRMT6/TRMT61A in cancer tissues was significantly higher than that in adjacent tissues, which was linearly correlated with the m1A levels in urine. As an m1A-modified methyltransferase, high expression of TRMT6/TRMT61A increased m1A modification and decreased apoptosis of tumor cells. Therefore, m1A can be used as an important biomarker for early bladder cancer.^158,159 A follow-up study revealed that high expression of TRMT6/TRMT61A in bladder cancer increased the levels of m1A modification at position 4 (corresponding to position 58 in mature tRNA; Figure 7A) in tRF-3b (tRNA-derived RNA fragment 3b). Interestingly, the m1A modification in tRFs negatively affects the gene silencing effect of tRFs. Therefore, with the increase of m1A4 levels, the gene silencing effect of tRF-3b was diminished on MBTPS1 and CREB3L2—molecules related to the unfolded protein response (UPR) signaling pathway—thereby promoting UPR and regulating tumor cell proliferation and apoptosis.²⁰

The higher the stage of gastrointestinal tumor, the higher the expression of TRMT6, TRMT61A, TRMT10C, ALKBH3, and YTHDF2 in patients, which regulate ErbB and mammalian target of rapamycin (mTOR) signaling pathway activity by dynamically regulating m1A levels, thereby affecting tumor growth invasion.¹⁶⁰ A recent study showed that alterations in m1A-related regulatory genes, such as TRMT6/TRMT61A, TRMT10C, and YTHDF1, were not only associated with the tumor-node-metastasis (TNM) stage of hepatocellular carcinoma (HCC) but were also found to be significantly associated with TP53 mutations. TP53 is a tumor suppressor gene that inhibits tumor cell proliferation, and the occurrence of TP53 mutations leads to uncontrolled tumor cell expansion.¹⁶¹ TRMT6/TRMT61A upregulation increases the m1A levels of tRNA in HCC stem cells, enhancing the translation of PPARδ, which initiates cholesterol synthesis, activates the Hedgehog signaling pathway, and ultimately promotes self-renewal and tumorigenesis of liver cancer stem cells.¹⁶² Another recent study reported that ALKBH3 expression was increased in HCC compared with that in paraneoplastic tissues, ALKBH3 expression levels were closely correlated with tumor differentiation and TNM stage, and patients with higher expression levels had a worse prognosis. ALKBH3 knockdown inhibits the proliferation of HCC cells in vitro and in vivo.¹⁶³ Possibly, like lung cancer cells,¹⁴⁶ ALKBH3 knockdown may play a tumor-suppressing role through p21/p27-mediated cell-cycle arrest in the G1 phase, indicating the functional role of m1A modification in the cell cycle. Taken together, poor prognosis was observed in HCC patients with elevated levels of TRMT6/TRMT61A or ALKBH3 expression, which may play a role in promoting cancer by regulating m1A methylation levels in different pathways, suggesting that dysfunction in m1A methylation plays a key role in HCC. Based on the above studies, a recent study by Wang et al. suggests that the TRMT6/TRMT61A complex may be a potential target for HCC treatment. Based on the screening of over 1,600 known drugs, it was found that thiram can effectively block the interaction of TRMT6 and TRMT61A, thereby significantly inhibiting the proliferation of HCC cells. Thiram treatment significantly reduced m1A levels in HCC cells without affecting levels of other RNA modifications (including m1G and Ψ), indicating that the anti-proliferative effect of thiram was significantly associated with a reduction in m1A levels in cells. In mice, the combination of thiram and a PPARδ antagonist (GSK3787) enhanced the tumor-suppressing effect and improved the survival rate.¹⁶²

Several studies have suggested that increased TRMT6/TRMT61A activity contributes to the maintenance of high tRNA_i^Met levels, which promotes the synthesis of tumor-associated proteins by enhancing translation levels, ultimately leading to malignant transformation in cells or cancer progression. For instance, in gliomas, TRMT6/TRMT61A is significantly expressed, which promotes glioma cell proliferation and reduces apoptosis by increasing tRNA_i^Met stability. Recent studies have revealed that PKCα can bind to TRMT61A in the cytoplasm and affect the nuclear translocation of TRMT61A, thereby regulating the formation of the TRMT6/TRMT61A complex and preventing the formation of m1A58 modifications in tRNA. Therefore, TRMT6/TRMT61A activity can be strictly controlled by regulating PKCα to prevent translation dysregulation that favors tumor development.¹⁶⁴

Clinical significance of m1A

Numerous studies have shown that m1A is important for the diagnosis and treatment of various diseases. In various disease states, m1A levels are altered to varying degrees in human body fluids. Parsons et al.¹⁶⁵ analyzed urine samples from 62 patients with interstitial cystitis (IC) and 33 controls and found that the m1A level in the urine of patients with IC was increased by 51%. As a urinary toxic cation, m1A binds to the anions of glycosaminoglycan (GAG) layer on the surface of the bladder mucosa and disrupts the GAG layer, thereby impairing its ability to regulate epithelial permeability and increasing the risk of IC. The research also showed that Tamm-Horsfall protein (TPH) in urine is negatively charged, which can bind to m1A and neutralize its toxicity, thereby exerting a protective effect on the bladder mucosa. Elevated m1A has been found in urine¹⁶⁶ or serum¹⁰⁹ samples from patients with gestational diabetes mellitus (GDM); thus, the diagnosis of GDM can be improved by detecting m1A. Furman et al.¹⁶⁷ found that increased m1A and ac4C levels in mouse blood were associated with hypertension in mice. m1A, in combination with ac4C, promotes the expression of the NLRC4 gene, initiates and activates the NLRC4 inflammasome, induces the production of IL-1β, and further activates platelets and leukocytes, thereby increasing blood pressure. The elevated expression of inflammasome gene modules is associated with all-cause mortality in older individuals over 85 years of age. In addition, researchers found that the urinary m1A in undialyzed patients with chronic renal failure was significantly reduced, as the reduced excretion of m1A led to its abnormal accumulation in the serum of uremic patients, which indicated that RNA metabolism was altered in uremic patients.¹⁶⁸

The level of modified nucleosides excreted in human urine has been demonstrated to be associated with the progression of several cancers.^18,19 RNA, especially tRNA, is the major source of modified nucleosides in urine. After tRNA metabolism, the unmodified nucleosides can be reused for nucleic acid synthesis either directly or after degradation by the salvage pathway, whereas modified nucleosides cannot be recovered due to changes in the stereostructure and are all excreted in the urine.¹⁸ Thus, the level of modified nucleosides in urine reflects RNA degradation in the organism. In 1966, Mandel et al.¹⁶⁹ first reported that the content of modified nucleosides was higher in cancer cells than in normal cells. In cancer cells, the metabolic turnover of tRNA is promoted,¹⁷⁰ and, because m1A and ψ are widely distributed in tRNA, massive excretion of m1A can be observed in the urine of cancer patients. For example, elevated levels of various uridine metabolites (including m1A) have been found in urine samples from patients with urogenital cancer (UC), indicating the diagnostic power of m1A for UC and as a reliable and convenient tool for UC diagnosis.¹⁷¹ Thomale et al.¹⁷² measured the urinary excretion rate of 12 modified nucleosides (including m1A and ψ) in mice by injecting a single dose of 3-methylcholanthrene. They found that mice with advanced tumors excreted several-fold higher levels of nucleoside metabolites than normal mice. The excretion rate of m1A was significantly increased before tumor diagnosis, whereas the excretion rate was not significantly changed in control mice and mice injected with carcinogens but without tumor formation. Significant upregulation of m1A was observed in the urine of individuals with women’s cancers, including breast, ovarian, and cervical cancer.¹⁰⁸ The combined detection of m1A in urine with serum CA153 significantly improved the diagnostic sensitivity of breast cancer and can be used for the early diagnosis of breast cancer.¹⁷³

In addition to the early diagnosis of the disease, m1A can also be used to infer the long-term prognosis of the disease. Compared with that in patients with early breast cancer, the level of m1A in the urine of patients with advanced stages increased more significantly, and the higher the m1A excretion, the lower the 5-year survival rate of patients, thereby indicating that the high urinary excretion of m1A in breast cancer patients has unfavorable prognostic significance.¹⁷⁴ In addition, m1A excretion in urine was higher in patients with bone metastases compared with that in other sites of cancer metastasis.

The above results indicate that, although the increase of m1A in urine does not imply the occurrence of a specific disease (non-specific marker), the detection system still has certain advantages; that is, compared with serum or internal organs of the body (such as liver, lung, and bone marrow), urine is very easy to collect, and the detection method is simple and rapid and can determine and process numerous samples in a short time. Therefore, the detection of m1A in urine is still a rapid and effective means of disease screening or monitoring.

Conclusions and future directions

In this review, we summarize the discovery and distribution of m1A modifications in RNA, m1A-related regulators, current methods for detecting m1A modifications, the biological function of m1A, the clinical significance of m1A, and, most importantly, the development of m1A in various diseases, especially the potential role of m1A in cancer.

The m1A58 modification in tRNA is considerably conserved in bacteria, Archaea, and eukaryotes, especially in tRNA_i^Met. m1A9 and m1A58 are essential for tRNA thermostability and translation initiation.^41,42 In human cells, both m1A1322 in cytoplasmic rRNA and m1A947 in 16S mt-rRNA are highly m1A-modified sites that affect ribosome biogenesis, inhibit the formation of translation initiation complexes, and mediate cellular resistance to antibiotics and sensitivity to oxidative stress.¹¹ m1A modifications in mRNA have different biological functions: depending on their location in different regions, they either promote translation by stabilizing the mRNA structure or inhibit translation or cause translation disruption by inhibiting base pairing with tRNA.

Writers catalyze the installation of m1A in RNA, which are either specific or non-specific. For instance, NML is only responsible for the installation of m1A1322 modification in human 28S rRNA, whereas TRMT61B catalyzes the formation of m1A in mt-tRNA,⁵⁸ mt-rRNA,³² and mt-mRNA¹³ simultaneously. Erasers are responsible for erasing m1A modifications, which can maintain genomic stability by reducing DNA alkylation damage or have transcriptional regulatory functions by regulating transcriptome m1A levels. Finally, readers recognize m1A methylation, which affects RNA splicing, degradation, translation, nuclear export, and other biological processes. However, cellular proteins involved in binding to m1A-modified mRNA as readers remain to be explored, and how the key target transcripts of these m1A readers affect the cellular activity and the underlying pathways and mechanisms mediating these changes is still far from being elucidated.

m1A and its related regulators play various important roles in human diseases. Deletion of NML, TRMT10C, or TRMT6/TRMT61A leads to embryonic lethality to varying degrees, either due to impaired erythropoiesis, mitochondrial dysfunction, or severe deficiencies in protein production.^{14,15,128,130} When tissues or cells encounter different stress stimuli, the level of m1A modification within the RNA is dynamically altered to adapt to their own growth needs. When stress occurs, m1A-modified mRNAs promote the formation of SGs and accelerate the assembly of polyribosomes and the translation process when stress is relieved.¹⁷ Considering that different cancers have different genetic backgrounds, m1A modifications in RNA can regulate oncogene expression (such as RNA splicing and degradation), cancer stem cell pluripotency, cell proliferation, differentiation, migration, angiogenesis, and the tumor microenvironment to control cancer progression. Interestingly, m1A appears to act as a double-edged sword in cancer treatment. High m1A modification levels in some tumors promotes tumor progression, whereas m1A removal in other tumors promotes tumor progression. For instance, TRMT6/TRMT61A upregulation increases the m1A level in HCC, which plays a tumor-promoting role by activating the Hedgehog signaling pathway, whereas, in certain cancers, ALKBH3 exerts a tumor-promoting role by erasing m1A modifications in tRNA.^67,152 In addition, the same m1A-related regulators may exert different biological functions through different mechanisms in the same cancer. For example, in certain breast cancers, ALKBH3 was found to stabilize and increase the half-life of CSF-1 mRNA by erasing m1A modifications, thereby increasing cancer cell invasion.¹⁵¹ In another scenario of breast cancer, the CpG island in the promoter region of ALKBH3 gene was highly methylated, resulting in silencing of ALKBH3 transcription and increased DNA alkylation damage, thereby promoting the occurrence and development of tumors and ultimately leading to a significant decrease in patient survival.¹⁵⁴ Aberrant expression of ALKBH3 has been observed in numerous cancer tissues; however, considering that it also has an alkylation repair function and is involved in maintaining normal DNA structure, whether ALKBH3 plays a role in tumor development by regulating m1A levels still needs to be verified by future experiments. Overall, these studies suggest that the complex regulatory network of m1A methylation is active in diverse cancers and remains to be further explored. In addition, some researchers are focusing on targeted cancer therapies associated with m1A, such as treatment with HUHS015, which significantly inhibits proliferation of androgen-independent prostate cancer cells by inhibiting ALKBH3 activity, thereby exerting an antitumor effect.^175,176,177 Finally, there are still few studies on m1A modification in lncRNAs in cancer, with only a few reports on colorectal and head and neck cancers.^31,178

The excretion of m1A in human urine is closely associated with the progression of various diseases. Urinary m1A is mainly derived from the degradation of tRNA. Therefore, any malignant disease, such as cancer, can alter the urinary m1A excretion levels by affecting RNA catabolism. Therefore, the quantification of m1A in urine can be used for early diagnosis of diseases and for monitoring the evolution of diseases. In addition, further large-scale clinical studies are required to confirm the specificity and sensitivity of m1A in disease diagnosis and prognosis.

Epitranscriptomics is an important subfield of epigenetics that can help unravel the biological mechanisms of disease development and may provide new targets for disease therapy. Although m1A has been the focus of many recent studies, full understanding of the mechanism is still very far away. However, we believe that future research on m1A modification will focus on the following aspects: first, finding more m1A-related reading proteins and revealing the molecular mechanisms by which the target transcripts of m1A reading proteins mediate downstream biological functions; second, constructing models of complex regulatory networks of m1A and its related regulators in diseases; third, advancing the application of m1A for the early diagnosis and clinical prognosis of diseases; and fourth, developing more effective drugs related to m1A to provide new therapeutic strategies for cancer treatment.