Explore the roles of m6A in RNA and its writers, erasers, and readers in RNA metabolism and find reliable sequencing to study these targets.
Recommended Reading: Epitranscriptomic Modifications and How to Detect Them.
What is m6A Modification?
Eukaryotic RNA contains over 160 modifications, primarily found on tRNAs and rRNAs. These modifications include m1A, m6A, m7G, m5C, and others. However, due to technological limitations, only a few types of modifications, such as "m6A (N6-methyladenosine)" and m5C, have been extensively studied, with m6A being the most researched. m6A refers to the addition of a methyl group to the N atom at the 6th position of adenosine.
In plants, m6A sites are widely distributed. Apart from directly modifying mRNA (impacting translation efficiency, RNA stability, alternative splicing, nuclear transport, etc.), m6A can also influence downstream gene expression by regulating non-coding RNAs. For example, it can regulate pri-mRNA processing, influence the binding of long non-coding RNAs (lncRNAs) to microRNAs (miRNAs), facilitate circular RNA (circRNA) translation, and affect the three-dimensional structure of lncRNAs when interacting with proteins. Among RNA modifications, RNA methylation accounts for approximately 60%, and m6A, being the earliest reported mRNA modification, represents the majority of methylation modifications, constituting up to 80% of the total.
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The Dynamic Regulation of m6A
The dynamic regulation of m6A involves three main categories of proteins: writers, erasers, and readers. Current research primarily focuses on understanding the specific functions and mechanisms of action of these proteins. Numerous studies have revealed that dysregulation of m6A-related proteins, such as writers, erasers, and readers, leads to abnormal m6A modifications in critical genes, resulting in post-transcriptional abnormalities in the expression of cancer-related genes.
m6A Writers
Writers, also known as methylation transferases, initiate m6A methylation. One significant example is METTL3, a writer that acts as an oncogene in AML cells. METTL3 can be recruited to chromatin by the CAATT-box binding protein CEBPZ, leading to increased m6A levels on mRNA transcripts like SP1 and SP2.
m6A Erasers
Erasers, or demethylation enzymes, remove m6A methylation modifications. FTO (ALKBH9), an eraser, can function as an oncogene, promoting leukemogenesis and inhibiting the differentiation of leukemic cells mediated by all-trans retinoic acid (ATRA). FTO reduces the abundance of m6A on mRNA transcripts such as ASB2 and RARA, thereby enhancing their stability.
m6A Readers
Readers are proteins that recognize m6A modification sites. Different reader proteins exhibit distinct effects on RNA modifications. Dysregulation of reader proteins can lead to misinterpretation of modified RNA and subsequent disruption of RNA metabolism. YTHDF2, the first reported m6A reader, has been extensively studied in various cancer types and demonstrated to have oncogenic properties across most cancer types.
Reversible m6A modification on mRNA. (Huang et al., 2020)
The dynamic regulation of mRNA m6A modification in plants involves writer and reader proteins, which recognize and control various aspects of plant development, including embryonic development, morphological differentiation, root development, floral organ development, and response to stress. Similarly, maintaining appropriate m6A levels and gene expression in human tissues and cells is crucial. Mutations or dysregulation in the writer and eraser proteins are often linked to diseases such as cancer. For instance, mutations in the critical domain region of METTL14 can disrupt the progression of endometrial cancer during advanced stages.
The Regulatory Function of m6A Modifications on mRNA
m6A modifications are added to RNA transcripts during the transcription process and play a significant role in gene expression after transcription. They bring about changes in the structure of RNA or alter its recognition by m6A-binding proteins, also called readers. While the amino acid coding remains the same between m6A-modified and unmodified codons during translational elongation, the presence of m6A modifications on mRNA acts as an obstacle to delayed tRNA regulation, resulting in the disruption of translational elongation kinetics.
Functions of m6A modifications on coding RNAs. (Huang et al., 2020)
m6A Modifications on Non-Coding RNAs
m6A modifications have been discovered not only in protein-coding mRNAs but also in non-coding RNAs (ncRNAs) such as lncRNAs, circRNAs, miRNAs, snRNAs, and others. These modifications have proven to be crucial for the expression and functionality of these ncRNAs.
The impact of m6A modifications on lncRNAs is particularly noteworthy, as they can affect RNA-protein interactions. For instance, MALAT1, a highly conserved star lncRNA, has consistently been associated with tumorigenesis and metastasis when it undergoes mutation or up-regulation. Additionally, m6A modifications play a role in RNA-RNA interactions of lncRNAs. In mouse embryo development, for instance, the internal m6A modification of the large intergenic coding RNA 1281 (linc1281) is essential to block stem cell pluripotency-associated let-7 family miRNAs and ensure proper development. Conversely, lncRNAs can also interact with m6A regulators to enhance their functionality.
A prevalent m6A motif, GGAC, is highly abundant on pri-miRNAs but absent on pre-miRNAs and mature miRNAs. Pri-miRNAs with m6A modifications can be recognized by hnRNPA2B1, which interacts with DGCR8 and facilitates miRNA processing.
The distribution of m6A enrichment in circRNAs differs from that observed in mRNAs. Instead of being dispersed throughout the transcript, m6A modifications in circRNAs tend to concentrate at the translation initiation sites of their corresponding mRNAs. These m6A modifications in circRNAs can also contribute to the translation of circRNA-encoded proteins by recruiting YTHDF3 and initiation factor eIF4G2.
Functions of m6A modifications on non-coding RNAs. (Huang et al., 2020)
m6A Modifications in Cancer Research
M6A modifications within tumors possess a broad spectrum of effects on gene expression by interacting with a diverse array of reader proteins and associated complexes. These modifications, despite not directly altering base pairing and coding function, impact critical processes like embryonic and hematopoietic stem cell self-renewal and differentiation, tissue development, circadian rhythms, heat shock or DNA damage response, and sex determination. Recent findings indicate that dysregulation of both the overall m6A modification level and the expression of regulatory proteins involved in its writing, erasing, and reading (Writers/Erasers/Readers) is prevalent in various cancer types. This dysregulation plays a pivotal role in the initiation, progression, metastasis, drug resistance, and recurrence of cancer. Additionally, other intracellular events, such as m6A site mutations, and extracellular stimuli can also influence cellular m6A modifications.
Deregulation of m6A modifiers in human cancers. (Huang et al., 2020)
Detection Methods for m6A RNA Methylation Modification
Various techniques are employed for detecting m6A RNA methylation modification, such as LC-MS/MS, colorimetric methods, miCLIP-seq, MeRIP-seq, and more. Currently, high-throughput sequencing-based analytical methods play a crucial role in localizing and analyzing RNA modifications.
Refer to our article Overview of Sequencing Methods for RNA m6A Profiling for more details.
Immunoprecipitation-Sequencing Analysis
To achieve specific detection of m6A, immunoprecipitation coupled with high-throughput sequencing is utilized. This approach involves pre-enrichment of m6A-containing RNA fragments using m6A-specific antibodies, resulting in m6A-specific methylated RNA immunoprecipitation coupled with next-generation sequencing (MeRIP-seq/m6A-seq). This enables the identification and analysis of m6A sites.
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Improved Methods for Single-Base Resolution Detection
To achieve single-base resolution detection of m6A methylation, optimized methods have been developed based on MeRIP-seq. These methods include m6A-cross-linking immunoprecipitation (m6A-CLIP) and m6A individual-nucleotide-resolution cross-linking and immunoprecipitation (miCLIP). By utilizing 254 nm UV irradiation, covalent cross-links are induced between the antibody and m6A-containing RNA. During reverse transcription, the antibody-RNA cross-linking site causes cDNA mutation or premature termination, enabling the identification of m6A sites with high precision through high-throughput sequencing.
Photo-Crosslinking Assisted m6A Sequencing
Another method, PA-m6A-seq (Photo-crosslinking-assisted m6A sequencing), was developed by Chen et al. This method incorporates a photo-reactive nucleoside analog, s4U, which enhances cross-linking efficiency. The cell culture medium is supplemented with s4U, and after immunoprecipitation, the antibody and m6A-containing molecules are incubated under 365 nm UV irradiation. Covalent cross-links are formed between the antibody and m6A-containing RNA, and the bound s4U leads to a T-to-C transition at the cross-linking site. This approach enables the recognition of m6A modification sites with single-base resolution.
Single Molecule Sequencing
Long-read sequencing technologies, such as single-molecule real-time (SMRT) sequencing and nanopore sequencing, allow direct localization of RNA modifications without sample amplification. In RT-SMRT analysis, the kinetic signal of reverse transcriptase passing through m6A differs significantly from that of A in control RNA. Moreover, the pulse frequency at the m6A position is noticeably lower compared to that of natural A. These techniques enable direct localization and analysis of not only m6A but also various other RNA modifications. Nanopore sequencing, in particular, characterizes current monitoring to differentiate the four nucleobases and distinguish modified nucleobases like I, m6A, and 5-mrC in RNA.
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Reference:
- Huang, Huilin, Hengyou Weng, and Jianjun Chen. "m6A modification in coding and non-coding RNAs: roles and therapeutic implications in cancer." Cancer cell 37.3 (2020): 270-288.
