RNA Modifications in Humans

In recent years, with the development of RNA modification detection technologies such as high-throughput sequencing, our understanding of RNA modification has progressed greatly. Common types of RNA modifications include methylation, adenylation, RNA editing, etc. RNA modifications are distributed on various types of RNAs in cells. RNA modifications play an important role in human physiological processes, which can affect RNA stability, cellular gene expression and protein function. A growing number of studies have shown that aberrant RNA modifications are associated with the development of various diseases such as cancer and neurodegenerative diseases, and RNA modifications have the potential to provide new directions and targets for disease diagnosis and treatment. In this article, we will detail the relationship between RNA modifications and humans.

Cellular stress response

RNA modification plays an important role in the cellular stress response by affecting the gene expression levels of modified RNAs and signaling cascades in the cell to regulate cellular adaptation to unfavorable environments that include oxidative stress, metabolic stress, heat shock, DNA damage, and endoplasmic reticulum stress.

Oxidative stress

Oxidative stress causes damage to cells and tissues and is closely related to many diseases such as diabetes and heart disease.

• Studies have shown that in mouse renal tubular epithelial cells, the m6A-modifying enzyme METTL3 can increase the m6A-modification site on miR-873-5p, and the methylated miR-873-5p can be recognized by miRNA processing complex DGCR8 and induced into mature RNA, which can inhibit the KEAP1 gene and activate the NRF2-mediated antioxidant pathway, enabling mouse renal tubular epithelial cells to cope with mucin-induced oxidative stress. mediated antioxidant pathway, enabling mouse renal tubular epithelial cells to cope with mucin-induced oxidative stress (Wang J et al., 2019).
• In colon cancer cell lines and HeLa cells, NSUN2 was able to increase the m5C methylation site on p21 mRNA to enhance the translation efficiency of p21 mRNA, and METTL3/METTL14 was also able to increase the m6A methylation modification site on p21 mRNA to further enhance the expression of p21. This RNA modification co-regulatory mechanism plays an important role in cell cycle regulation and cellular stress responses (Li Q et al., 2017).
• In human glioma, research has found that the absence of NSUN5 leads to low methylation of the 28S rRNA, resulting in a general reduction in protein translation and weakened ability to respond to oxidative stress. Although overall protein translation levels are reduced, this low methylation state promotes the translation of important proteins that are adapted to oxidative stress, especially the expression of the antioxidant NQO1, which indicates that RNA modification can adjust the translation program of tumor cells to respond to oxidative stress environments (Wilkinson E et al., 2021).

Heat shock

• The researchers found that m7G is enriched in the 5'UTR region and in AG-rich environments, a feature conserved across different human/mouse cell lines and mouse tissues. Strikingly, the internal m7G modification was dynamically regulated under both H2O2 and heat shock treatments, with significant accumulation in the CDS and 3'UTR regions and a role in promoting mRNA translation efficiency.
• It was found that the 5'UTR region of newly transcribed mRNAs was enriched with m6A modification under heat shock stress.YTHDF2 was induced to localize to the nucleus by stress to regulate the dynamic methylation of 5'UTR. YTHDF2 in the nucleus also restricted the activity of the m6A demethylase FTO to maintain the 5'UTR methylation level of stress-induced transcripts (Zhou J et al., 2015).

DNA damage

The DNA damage response (DDR) is a system for detecting and repairing damaged DNA to prevent cells from dividing before repair is complete.

• Upon exposure to ultraviolet (UV) radiation and DNA double-strand breaks, METTL3 rapidly localizes to DNA damage sites and enriches m6A methylation modifications on DNA damage-associated RNAs, thereby increasing the accumulation of DNA-RNA hybrids, and METTL3 also recruits DNA damage-associated proteins (e.g., YTHDC1, RAD51, and BRCA) to initiate a homologous recombination reaction to repair damaged DNA. In the absence of METTL3 catalytic activity, cells showed increased UV sensitivity and delayed repair of cyclobutane pyrimidine (CPD) adducts, further demonstrating the importance of m6A in the UV response and DNA damage repair.
• After exposure to UV radiation resulting in NA damage, a variety of DNA polymerases are required to rapidly aggregate at the damage site to repair the damaged DNA, such as the DNA polymerase Pol κ. The catalytic activity of METTL3 is required for Pol κ to localize to the site of UV-induced DNA damage, and overexpression of Pol κ significantly inhibited the defective removal of CPDs associated with METTL3 deletion (Xiang Y et al., 2017).

Cell proliferation and differentiation

• It was found that METTL3 enhances the translation efficiency of PHLPP2 mRNA by enriching the m6A modification site on PHLPP2 gene mRNA, leading to an increase in the synthesis of PHLPP2 protein thereby inhibiting the proliferation of endometrial cancer cells. Knockdown of Mettl3 decreases this m6A-dependent translation of the PHLPP2 gene decreasing its protein synthesis efficiency and leading to increased proliferation of endometrial cancer cells(Parial R et al., 2021).
• METTL3 is an essential m6A-regulated enzyme in colorectal carcinogenesis. METTL3 promotes the translation of GLUT1 in an m6A-dependent manner, which in turn activates the mTORC1 signaling pathway and facilitates the initiation and progression of CRC; therefore, knockdown of METTL3 inhibits colorectal carcinogenesis, and inhibition of the mTORC1 gene expression further enhances the anti cancer effect of silencing METTL3 gene in anticancer effects in CRC(Chen H et al., 2021).
• In leukemia, FTO promotes acute myeloid leukemia (AML) cell proliferation by decreasing the m6A levels of the 3'-UTR of Asb2 as well as the 3'- and 5'-UTRs of Lara, leading to reduced ASB2 and RARA protein expression. In addition, YTHDF2 enhances leukemia cell proliferation by promoting m6A-dependent mRNA degradation of Wee1. PRRC2A is an m6A-binding protein that binds and stabilizes Olig2 mRNA in an m6A-dependent manner, thereby promoting oligodendrocyte proliferation (Li Z et al., 2017).
• It was shown that embryonic stem cells (ESC) exhibit increased pluripotency but reduced self-renewal capacity after knockdown of the m6A-modified methyltransferase Mettl3 gene, and that EpiSC also show a stronger tendency to differentiate after the reduction in m6A levels. Furthermore, deletion of Ythdf3 in ESC resulted in a loss of pluripotency and accelerated cardiac differentiation by promoting the expression of cardiomyocyte-specific genes, and also showed an increase in cell proliferation. In contrast, deletion of Ythdf1 results in significantly impaired cardiomyocyte differentiation and a slight decrease in proliferation.
• During pluripotent stem cell differentiation, Mettl3 deficiency significantly inhibits the self-renewal capacity of induced pluripotent stem cells (piPSCs) and promotes their differentiation. Meanwhile, YTHDF2 inhibits induced pluripotent stem cell differentiation by reducing the stability of m6A-modified transcripts associated with neural development.
• It was found that m6A RNA methylation was directly involved in the regulation of myogenic differentiation. Overexpression of Mettl3 positively regulates m6A RNA methylation and further promotes myogenic differentiation. Knockdown of Mettl3 resulted in decreased mRNA expression levels of the myogenic transcription factor MyoD. On the other hand, the demethylase FTO significantly promotes myogenic differentiation. In myoblasts of female embryos, FTO promotes cell proliferation and myoblast differentiation through the adhesion site pathway (Bo Wei et al., 2022).

Apoptosis and senescence

• METTL3 protein acts on cell survival-related genes, such as BCL2, to increase the level of m6A modification on the genes, and then significantly increase their gene expression levels to inhibit apoptosis. Moreover, it has been shown that the upregulation of c-Myc expression may be related to the increase of m6A modification on its mRNA, and the upregulation of c-Myc expression can enhance the cascade response of signaling pathways related to cell proliferation, thus inhibiting cell apoptosis.
• The m6A modification promotes the expression and enhances the protein activity of AKT pathway genes and can inhibit apoptosis by increasing the mRNA stability of certain genes in the AKT pathway. Conversely, removal of m6A modifications may result in degradation of AKT pathway-associated mRNAs, which may reduce AKT signaling activity and increase the risk of apoptosis. For example, inhibition of m6A-modification-related enzymes (e.g., METTL3) leads to a decrease in the stability of AKT-associated mRNAs, and this regulatory mechanism is further supported by the fact that researchers have observed that there is indeed an interaction between the m6A modification and the AKT-associated mRNAs by using experimental methods such as RNA immunoprecipitation (RIP) (Liu BH et al.,2021).
• Several studies have revealed a link between m6A methylation and cellular senescence. Using human cervical cancer HeLa cells, researchers found that METTL3/METTL14 protease-enhanced m6A methylation levels of p21 mRNA increased the accumulation of p21 protein in HeLa cells, but there was no significant change in the total level of p21 mRNA, suggesting that m6A methylation enhances the translational efficiency of p21 protein. When HCT116 cells were exposed to hydrogen peroxide, the levels of p21 and METTL3/METTL14 proteins were significantly increased, and senescence-associated β-galactosidase ((SA)-β-gal) activity was also enhanced, suggesting an important role for METTL3/METTL14 in oxidative damage-induced senescence (Li Q et al.,2017).
• Some studies have also revealed N6-methyladenosine (m6A) RNA modification profiles in peripheral blood mononuclear cells (PBMCs) from young and elderly populations. The results indicate that the overall level of m6A methylation is reduced in the aging population, and in particular, the methylation modification of protein-coding mRNAs is most predominantly reduced. Transcripts modified with m6A were generally more highly expressed than unmodified transcripts. Among the many methylated mRNAs, mRNAs for DROSHA and AGO2 were heavily modified in young PBMC, which is consistent with the decrease in steady-state levels of AGO2 mRNA in aged PBMC. In addition, the down-regulation of AGO2 in proliferating human diploid fibroblasts (HDFs) was also associated with its mRNA modification and reduced steady-state levels. In HDFs, overexpression of RNA methyltransferases stabilized AGO2 mRNA but not DROSHA and DICER1 mRNA. miRNA abundance was also altered in young and old PBMCs, which may be associated with AGO2 expression. Taken together, we reveal the effect of mRNA methylation on AGO2 mRNA abundance, which in turn inhibits miRNA expression during human aging (Casella G et al., 2019).

Metabolic disease

• Several recent studies have revealed a key role for m6A modifications in the pathogenesis of type 2 diabetes (T2D). elevated blood glucose levels in T2D patients affect the dynamic regulation of m6A levels in cells.Elevated blood glucose levels in T2D patients affect the dynamic regulation of m6A levels in cells. Decreased expression of the demethylase FTO mRNA and increased expression of the methyltransferases METTL3, METTL14, and WTAP lead to increased cellular blood glucose levels.As these m6A modifying enzymes lead to increased m6A modifications in T2D patients thereby affecting β-cell survival and insulin secretion, T2D patients are unable to lower blood glucose levels. In addition, METTL3 inhibits hepatic insulin sensitivity by modifying FASN mRNA via m6A and promoting fatty acid metabolism.
• Low METTL3 activity in cell cultures reduces the m6A modification of the peroxisome proliferator-activated receptor (PPAR)α gene, leading to increased PPARα mRNA expression and extended transcript lifespan, which helps decrease lipid accumulation. Analysis indicates that YTHDF2 binds to PPARα mRNA, enhancing its stability and regulating lipid metabolism. In addition, zinc finger protein 217 (Zfp217) interacts with YTHDF2, activates the transcription of m6A demethylase FTO, promotes its binding to m6A sites on various mRNAs to reduce m6A modification, and promotes adipose differentiation (Zhang Y et al., 2021).

RNA modifications and their links to human disease(Jonkhout N et al.,2017)

Cancer

• NAT10-deficient cancer cells exhibit reduced cell growth and proliferation, which may be associated with reduced ac4C modification of FA metabolism-related genes, which in turn leads to decreased lipid levels. Studies have shown that cell proliferation is mainly dependent on FA metabolism genes, but in cancer cells knockdown of NAT10 gene leads to FA metabolism dysfunction, which eventually leads to cancer cell death.The expression of NAT10 is significantly up-regulated in cervical cancer cells, and the knockdown of NAT10 gene can inhibit cervical cancer cell proliferation, invasion, and migration.The main target of NAT10 in cervical cancer cells is the Heterogeneous Nuclear Ribbon Protein U-like 1 (HNRNPUL1). ribonucleoprotein U-like 1 (HNRNPUL1), and the NAT10 gene catalyzes the formation of RNA-modified ac4C, which enhances the stability of HNRNPUL1 mRNA and regulates the expression of HNRNPUL1, which in turn promotes the development of cervical cancer(Long Y et al.,2023).
• m6A modifications play an important role in the development and progression of many malignant tumors, especially in cancer types such as acute myelogenous leukemia (AML), hepatocellular carcinoma (HCC), colorectal cancer (CRC), non-small-cell lung cancer (NSCLC), and gastric cancer, which show significant expression abnormalities. Specifically, FTO, whose expression is significantly elevated in AML, can play a role as a proto-oncogene by decreasing the m6A levels of anchor protein repeat sequences and ASB2 and RARA mRNAs, thereby promoting the growth and differentiation of AML cells.The up-regulation of METTL14 increases the m6A levels of oncogenes, such as MYB and MYC, and enhances their stability and translational capacity, which in turn maintains leukemia stem cell self-renewal.YTHDF2 maintains the functional integrity of leukemia stem cells and promotes the proliferation of hematopoietic stem cells by decreasing the half-life of various m6A-modified mRNAs such as TNFR2 (Emma Wilkinson et al.,2022).
• The m1A (N1-methyladenosine) modification shows significant changes in modification levels in a variety of malignant tumors especially in cancers such as ovarian cancer, esophageal squamous carcinoma, pancreatic cancer, glioma and renal cell carcinoma. Studies have shown that these changes are closely related to tumor progression and prognosis, suggesting that m1A modifications may play an important role in the biological process of tumors. For example, ALKBH1 promotes tumor metastasis by regulating the m1A modification on SMAD7 mRNA, leading to a decrease in the expression level of SMAD7 protein. Moreover, the m1A modification-associated enzyme ALKBH1 showed high expression in colorectal cancer (CRC) tissues, and this high expression may affect the RNA modification levels of other RNA modifying enzymes (e.g., the m6A modifying enzyme METTL3), thereby promoting tumor development. This suggests that m1A modification may play a key role in signaling and metastasis of tumor cells (Li J et al.,2022).
• In the study of hepatocellular carcinoma (HCC), m5C modification plays an important role in the occurrence and development of HCC. By analyzing the m5C modifications of mRNAs in hepatocellular carcinoma tissues, 125 mRNAs were found to show up-regulation and 38 mRNAs showed down-regulation in cancer tissues, indicating that m5C modifications play a key role in the development of hepatocellular carcinoma tissues. In addition, NSUN2 and ALYREF improved the stability of YAP by increasing the m5C modification in the 3'UTR region of the YAP gene mRNA, and YAP could affect lung adenocarcinoma by acting together with the transcription factors Mycn and SOX10 to enhance the secretion of cellular exosomes (Song D et al.,2023).

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