Overview of Epitranscriptomics

Unraveling the mechanisms by which genetic variation translates into phenotypic diversity at the cell, tissue, or organism level has long been a major challenge in the field of biology. Gene expression is a complex biological process that bridges the gap between genotype and phenotype. However, studies such as gene mutations, gene regulation, epigenetics, and epitranscriptomics have shown that there is a crucial relationship between gene expression and disease. In recent years, scientists have begun to explore new ways to solve diseases from the perspective of RNA modification, i.e., epitranscriptomics.

What is Epitranscriptomics

Epitranscriptomics is the field of studying RNA modifications and their role in the regulation of gene expression. Unlike traditional transcriptomics, which focuses on gene expression levels, epitranscriptomics emphasizes the effects of post-transcriptional modifications of RNA molecules (e.g., m6A, m5C, etc.) on cellular function and biological processes. These modifications can regulate the stability, translation efficiency, and degradation pathways of mRNA, thereby affecting protein synthesis and cell metabolic status.

In recent years, epitranscriptomics has received particular attention in disease research, especially in the fields of cancer, metabolic diseases, and neurological diseases. By revealing the dynamics of RNA modifications, scientists are able to better understand the pathogenesis of these diseases and explore new therapeutic targets and biomarkers. Developments in epitranscriptomics provide us with a completely new perspective to help understand complex biological processes and disease states.

Relationship Between Epitranscriptomics and Epigenetics

Epitranscriptomics (also known as "RNA epigenetics") is a cutting-edge research area that has emerged in recent years and focuses on dynamic reversible chemical modifications of RNA. There are more than 100 known post-transcriptional modifications, many of which play an important role in gene expression regulation in eukaryotes.

Some people may ask, is epitranscriptomics epigenetics?

Epigenetics is defined as changes in phenotype or gene expression caused by mechanisms that are not underlying DNA sequence alterations, such as histone and DNA modifications, that regulate the release of genetic information encoded in DNA without altering the underlying sequence.

Considering the different substrates on which they act, we cannot consider epitranscriptomics as epigenetics. However, given the phenotypic influences determined by gene activity, we can assert that epitranscriptomics, epigenetics, and epiproteomics are two sides of the same coin, all participating in the complex interconnected web of what we define as "cellular epitony."

Key RNA Modifications

So far, researchers have discovered more than 100 different kinds of chemical modifications on RNA, most of which are distributed in high-abundance non-coding RNAs, such as rRNA, tRNA and snRNA, and play an important role in maintaining the function of non-coding RNAs. But excitingly, more and more modifications are being discovered, precisely quantified, and localized on mRNA, including N6-methyladenine (m6A), N6,2-O-dimethyladenine (m6Am), 5-methylcytosine (m5C), hypoxanthine (I), pseudouracil (Ψ), N1-methyladenine (m1A), 2′-O-methylation (Nm), N4-acetylcytosine (ac4C), and N7methylguanine (m7G). In particular, with the new discovery of many mRNA-modifying enzymes (Writer), De-modifying Enzymes (Eraser) and modifying recognition proteins (Reader), the reversible changes and dynamic regulation of mRNA chemical modification have re-aroused the interest of researchers and attracted more and more attention.

The discovery of dynamic reversible modifications means that during the life cycle of mRNA, cells can add or remove these modifications to carry out more direct and transient gene expression regulation, and achieve rapid response to changes in the external environment.

• Epitranscriptomics m6a: (N6-methyladenosine) is one of the most abundant reversible modifications in messenger RNA (mRNA), which is formed by the addition of a methyl group by a specific methyltransferase to the nitrogen 6 position of adenosine, accounting for about 50% of the total post-transcriptional modifications. This modification dynamically regulates the metabolism of mRNA by enabling its processes of "writing" (methyltransferase), "reading" (binding protein), and "erasing" (demethylase) through different collections of proteins. The m6A modification is mainly enriched near the stop codon and the 3' untranslated region and is involved in various stages of the RNA life cycle, including transcription, processing, translation, and degradation. As an important research object in epitranscriptomics, m6A modification plays a key role in regulating gene expression, RNA stability, splicing and translation. Recent studies have found that the abnormal expression of m6A-related proteins (such as METTL3/14, WTAP, FTO, ALKBH5 and YTHDFs) in tumors can lead to m6A methylation dysregulation, thereby affecting the expression of oncogenes and tumor suppressor genes, and then participate in the occurrence and development of tumors, which is closely related to the prognosis of patients. In addition, it has not yet been conclusively determined whether there is a corresponding regulatory system for m6A or m6Am on other types of RNA. Therefore, it is of great significance to explore m6A modification and its regulatory mechanism to understand gene expression and its role in a variety of diseases.

To learn more, please refer to Overview of m6A RNA Methylation and Overview of Sequencing Methods for RNA m6A Profiling.

• 5-Methylcytosine: m5C(5-Methylcytosine) is an abundant RNA modification formed by a specific methyltransferase enzyme that adds a methyl group to the 5-position carbon of cytosine and has been reported to be present in a variety of RNA species, including cytoplasmic and mitochondrial ribosomal RNA (rRNA) and transfer RNA (tRNA), as well as messenger RNA (mRNA), enhancer RNA (eRNA), and many non-coding RNAs. In eukaryotes, C5 methylation of RNA cytosine is catalyzed by enzymes of the NOL1/NOP2/SUN domain (NSUN) family, as well as the DNA methyltransferase homolog DNMT2. m5C modification can enhance the stability of RNA molecules, thereby protecting them from degradation, but its introduction may alter the secondary structure of RNA, thereby affecting its function and interactions. In addition, m5C modification is involved in a variety of cellular processes, such as gene expression regulation, cell proliferation and differentiation. m5C modification plays a key role in a variety of biological processes, and its dysregulation has been implicated in a variety of diseases such as cancer, neurodegenerative diseases, etc. Therefore, in-depth study of the mechanism and function of m5C not only helps to understand the basic biological processes, but also provides new potential targets for the diagnosis and treatment of diseases.

Epitranscriptomic Regulation

Epitranscriptomic regulation refers to the complex process of controlling gene expression through the chemical modification of RNA molecules. These modifications not only affect RNA stability, localization, translation, and degradation, but also play a key role in multiple biological processes and cellular functions. Here's a closer look at the various aspects of this area:

• RNA modifications: RNA molecules can be chemically modified by the addition of specific methyltransferases and other modifying enzymes. These modifications, including 5-methylcytosine (m5C), N6-methyladenosine (m6A), pseudouridine, etc., can significantly alter the structure and function of RNA. For example, m6A modifications are considered to be the most common internal modifications, capable of influencing the secondary structure of RNA, which in turn affects its interaction with proteins and its biological functions.
• Effect on stability and degradation: Modified RNAs typically exhibit greater stability, which allows them to remain in cells for a longer period of time, increasing their expression levels. Certain modifications may label RNA for recognition by RNA degradation mechanisms, regulating the intracellular concentration of specific transcripts. This regulation is essential for cells to respond to environmental changes and maintain homeostasis.
• Impact on translation: Epitranscriptome modifications can affect the translation of mRNA into proteins. For example, m6A modification has been shown to promote ribosome recruitment, thereby enhancing translation efficiency. In addition, different modifications may modulate translation speed and precision by affecting the assembly of translation initiation complexes or by altering the spatial configuration of mRNA.
• Regulation of non-coding RNAs: The non-coding RNA class, including microRNAs (miRNAs) and long non-coding RNAs (lncRNAs), also play an important role in epitranscriptome regulation. These non-coding RNAs can interact with modified RNAs, affecting their stability and translation, and are involved in a network of post-transcriptional regulation, helping cells finely regulate gene expression.
• Role in cellular processes: Epitranscriptome regulation plays a crucial role in cell differentiation, stress response, and regulation of the immune system. For example, during development, specific patterns of RNA modification can guide cells towards specific differentiation pathways. In addition, the dynamics of the epitranscriptome can help cells quickly adapt to environmental changes under stressful conditions.
• Dynamic properties: The modification patterns of the epitranscriptome are dynamic, and they vary depending on external environmental signals, developmental timing, and cellular state. This flexibility highlights the importance of the epitranscriptome in adaptive gene regulation, enabling cells to efficiently regulate gene expression under different physiological and pathological conditions.

Epitranscriptomics in Disease

Epitranscriptomics has shown great potential in disease research, revealing disease mechanisms, discovering potential biomarkers, and developing new therapeutic targets. By analyzing the chemical modifications of RNA and how it changes in different disease states, researchers can better understand how cells respond to pathological states, which in turn can support early diagnosis and personalized medicine. In addition, the combination of epitranscriptome and other omics data can help build disease-related molecular networks and promote the development of new drugs and precision treatments. The continued development of this field will greatly contribute to the innovation of our understanding of complex diseases and treatment strategies.

Cancer Epitranscriptomics

• Cancer epitranscriptomics is a fast-growing field that focuses on RNA modifications and their implications in cancer biology.Studies have shown that m6A modifications can affect various aspects of cell behavior, including proliferation, migration, and drug resistance in cancer cells. For example, the presence of m6A can stabilize or unstabilize specific mRNAs, affecting the levels of oncogenes and tumor suppressor genes. Key players in this process are m6A methyltransferases, such as METTL3 and METTL14, which are responsible for the addition of the m6A marker, and demethylases, such as FTO and ALKBH5, which are responsible for removing the marker. Dysregulation of these enzymes has been implicated in a variety of cancers, highlighting the importance of m6A homeostasis in tumor development.
• In addition to m6A, m5C modifications also play a key role in cancer biology. Studies have shown that this modification is related to the stability and translation of mRNA, and that m5C levels may vary between different cancer types. Enzymes responsible for the addition and removal of m5C-labels, such as NSUN and TET proteins, have been identified as potential therapeutic targets. The interaction between m5C modification and cancer progression remains an active area of research with significant implications for understanding tumorigenesis and developing new therapeutic strategies.

Ribonucleotide RNA modifications known to be of relevance in cancer and their enzymatic machineries(Kundig et al., 2018).

Epitranscriptomics in Metabolic Disease

• In recent years, epitranscriptomics has attracted extensive attention in the study of metabolic diseases. These diseases, including obesity, diabetes, and fatty liver disease, often involve complex metabolic pathways and regulatory mechanisms, and RNA modification plays an important role in these processes.In the context of metabolic diseases, the most well-known RNA modifications include N6-methyladenosine (m6A), 5-methylcytosine (m5C), and gamma-glucose (m1A). These modifications not only affect RNA stability and translation efficiency, but may also regulate gene expression by affecting the splicing, transport, and degradation of RNA. For example, m6A modifications have been shown to be able to regulate the expression levels of genes associated with energy metabolism, thereby influencing the metabolic state and function of cells. In metabolic diseases such as obesity and type 2 diabetes, alterations in m6A are often associated with dysregulation of the insulin signaling pathway, suggesting a critical role in metabolic homeostasis.
• In addition, it was found that the expression profile of m5C modification in adipose tissue was closely related to the occurrence of metabolic diseases. m5C modification regulates the expression of genes involved in lipid metabolism, such as fatty acid synthase and triglyceride synthase. These findings suggest that the regulation of m5C modification of RNA may provide new targets for the intervention of metabolic diseases.

Techniques of Epitranscriptomics

The methods of epitranscriptome sequencing are used to study the chemical modifications of RNA molecules, revealing their roles in gene expression and cellular functions.

• MeRIP-Seq (m6A RNA Immunoprecipitation Sequencing): Utilizes antibodies to specifically enrich mRNA containing N6-methyladenosine (m6A) modifications, followed by high-throughput sequencing of the enriched RNA.Identifying and locating the distribution of m6A modifications across the genome.
• PAR-CLIP (Photoactivatable Crosslinking and Immunoprecipitation): Crosslinks RNA-protein complexes using ultraviolet light, then enriches target RNA with specific antibodies before sequencing.Analyzing the interactions between RNA and binding proteins, as well as the effects of RNA modifications.
• A-to-I Edit-Seq (Adenosine to Inosine Editing Sequencing): Identifies sites where adenosine (A) is edited to inosine (I) in RNA through high-throughput sequencing to detect these editing events. Studying the role of RNA editing in gene regulation.
• scRNA-seq (Single-Cell RNA Sequencing): Sequences RNA from individual cells to study the epitranscriptomic modifications within cells. Revealing heterogeneity among cells and their epitranscriptomic features in different states.
• Ribo-Seq (Ribosome Profiling Sequencing): Sequences mRNA fragments occupied by ribosomes to study RNA modifications during the translation process. Analyzing how post-translational modifications impact protein synthesis.

Epitranscriptomics has a wide range of applications, including cancer research, neuroscience, developmental biology, immunology, drug discovery, and agricultural science. In cancer research, it helps identify tumor-specific markers and potential targets; In neuroscience, mechanisms of neurodegenerative and psychiatric diseases can be revealed; In developmental biology, analyzing RNA modifications can help understand gene expression regulation. In addition, epitranscriptomics can also be used for molecular diagnosis, providing new ideas for early diagnosis and prognosis assessment of diseases. Overall, this field will play an increasingly important role in basic research and clinical applications as technology advances.

References:

1. Kyung, Kleiner "Mechanisms of epitranscriptomic gene regulation."Biopolymers.112.1(2021):e23403.
2. Primac, Penning , "Fuks Cancer epitranscriptomics in a nutshel."Curr Opin Genet Dev. 75(2022):101924.