New Research on RNA Methylation Modifications (m6A)

What is RNA Methylation?

RNA methylation modification refers to the process in which methyl groups are added to nucleotides under the mediation of RNA methyltransferases. m6A, a prevalent form of RNA methylation in eukaryotes, is one of the most common types of RNA methylation modifications, characterized by the addition of a methyl group to the N6 position of adenine. Abundant research has shown that m6A modification plays a crucial role in regulating RNA processing, splicing, nuclear export, translation, stability, and even phase separation, which is essential for the development of various human diseases. Besides its vital role in normal hematopoiesis, central nervous system development, and reproductive system development, aberrant m6A modification has also been implicated in various types of human cancers. Additionally, increasing evidence suggests that m6A modification is closely associated with the occurrence and progression of cardiovascular diseases such as cardiac hypertrophy, heart failure, ischemic heart disease, and pulmonary arterial hypertension. The level of m6A modification is a dynamic reversible process regulated by methyltransferases (writers) and demethylases (erasers). Methyltransferases exert their function through a complex composed of multiple proteins, including methyltransferase-like protein 3 (METTL3), METTL14, METTL16, RNA-binding motif protein 15 (RBM15) and its homolog RBM15B, zinc finger CCCH domain-containing protein 13 (ZC3H13), vir-like m6A methyltransferase associated protein (VIRMA, also known as KIAA1429), and Wilms tumor 1-associated protein (WTAP), which transfer methyl groups from the donor S-adenosyl methionine (SAM) to adenine. Among them, METTL3, METTL14, and WTAP are the core members of this complex.

Schematic diagram of RNA modification types.

Subsequently, m6A methylation is recognized by m6A reader proteins such as the YTH domain family proteins (YTHDF1, 2, 3), insulin-like growth factor 2 mRNA-binding proteins (IGF2BPs), and heterogeneous nuclear ribonucleoprotein (HNRNP) family proteins. The reversible process of m6A demethylation is executed by erasers such as fat mass and obesity-associated protein (FTO) and ALKB homolog 5 (ALKBH5).

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What is RNA Methylation Enzymes?

RNA methylation modifications account for over 60% of all RNA modifications, and N6-methyladenosine (m6A) is the most prevalent modification on mRNA and lncRNAs in higher organisms. Currently, m6A modifications have been found to occur on microRNAs, circRNAs, rRNAs, tRNAs, and snoRNAs. m6A modification primarily occurs on adenine within the RRACH sequence, and its function is determined by "writers," "erasers," and "readers." The "writer" refers to the methyltransferase complex, known components of which include METTL3, METTL14, WTAP, and KIAA1429. ALKBH5 and FTO act as demethylases (erasers) to reversibly remove methyl groups. m6A is recognized by m6A-binding proteins, with known "reader" proteins including YTH domain proteins (including YTHDF1, YTHDF2, YTHDF3, YTHDC1, and YTHDC2) and the heterogeneous nuclear ribonucleoprotein (HNRNP) family (HNRNPA2B1 and HNRNPC).

• METTL3 was initially identified as a component binding to SAM, and its deficiency leads to a reduction in m6A peaks in mouse embryonic stem cells, Hela cells, and HepG2 cells. METTL3 and its homolog METTL14 are located on nuclear speckles rich in splicing factors, indicating a potential correlation between m6A modification and RNA splicing. WTAP interacts with the METTL3-METTL14 dimer and co-localizes with them in nuclear speckles, influencing methylation efficiency and participating in mRNA splicing. KIAA1429, as a new subunit of the methyltransferase complex, is essential for the overall methylation process. FTO, a member of the ALKB family, as the first discovered demethylase, can affect the RNA-binding ability of the splicing factor SRSF2, thereby regulating pre-mRNA splicing. Abnormal regulation by FTO has been found to be associated with obesity, brain malformation, and growth retardation, suggesting an important regulatory role of m6A in these diseases.
• ALKBH5 is another member of the ALKB family discovered to have demethylation activity, and it co-localizes with nuclear speckles in an RNase A-sensitive manner. It can directly catalyze the removal of the methyl group from m6A-methylated adenosine, unlike the oxidative demethylation by FTO. Additionally, ALKBH5 and its demethylation activity affect the efficiency of nascent mRNA synthesis and splicing, and ALKBH5 knockout male mice exhibit abnormalities in spermatogenesis, possibly due to changes in gene expression related to spermatogenesis. The functional execution of m6A mRNA modification mainly occurs through two pathways: finely regulating the structure of methylated transcripts to prevent or induce protein-RNA interactions; or being directly recognized by m6A-binding proteins, triggering subsequent reactions.

A class of proteins containing YTH domain function has been identified as m6A-modified binding proteins. Among them, YTHDF1, YTHDF2, YTHDF3, YTHDC1, and YTHDC2 have been confirmed as m6A-binding proteins. YTHDF1 mainly affects the translation of m6A-modified genes, YTHDF2 mainly affects the degradation of m6A-modified genes, and YTHDC1 binds to m6A-modified genes to affect their splicing. HNRNPC is a rich nuclear RNA-binding protein involved in pre-mRNA processing, and studies have shown that HNRNPC regulates the abundance and selective splicing of target transcripts through m6A-RNA binding.

What is the Functions of RNA Methylation?

RNA m6A modification plays a significant role in the regulation of gene expression, and abnormalities in its regulatory mechanisms may be associated with human diseases or cancer. Currently, it has been found that m6A may affect sperm development (ALKBH5, METTL3, Ythdc2), development (METTL3, FTO, ALKBH5), immunity (METTL3), UV-induced DNA damage response (METTL3, FTO), tumor initiation (YTHDF2), or metastasis (METTL14), stem cell renewal (METTL14), adipocyte differentiation (FTO), circadian rhythm, cell development differentiation, cell division, and other life processes.

Hotspot Research on RNA Methylation

• The main focus is to study the functions of methyltransferases, demethylases, and reader proteins associated with m6A modification, thereby investigating the biological functions and mechanisms of m6A modification. Typically, by knocking out m6A enzyme molecules, researchers study the expression of downstream functional genes and the methylation status of m6A, thereby mediating the phenotype and functional characteristics of cells through the modulation of related gene abnormalities (such as alternative splicing, stability, translation, and miRNA regulation).
• Research also involves the construction of m6A modification maps and the study of its mechanisms. This is achieved by using m6A methylation sequencing techniques (such as MeRIP-Seq, miCLIP) to construct m6A modification profiles of disease cell models or affected tissues, analyzing m6A motifs, peak numbers, and distributions, as well as the characteristics of genes associated with peaks. Combined RNA-seq studies are conducted to explore the relationship between m6A methylation and gene expression.