RNA Modifications Overview: Types, Mechanisms, and Their Biological Impact

Since the discovery and documentation of RNA modifications in the 1950s, more than 170 RNA modifications have been identified to date. These RNA modifications are widely distributed on various types of RNAs, such as messenger RNA (mRNA), transporter RNA (tRNA), ribosomal RNA (rRNA), non-coding small RNAs, and long-stranded non-coding RNAs (lncRNAs). In most cases, RNA modifications are reversible, similar to DNA methylation, e.g., m6A methylation is reversible. rRNA modifications are catalyzed by "writers," "erasers," and "readers," respectively "RNA modifications can affect the typical structure of ribonucleic acid and bases, thus affecting RNA splicing, translation, degradation, and translation efficiency, etc. Studies have shown that RNA modifications are associated with the onset and progression of many human diseases.

The Importance of RNA Modification

Regulates gene expression

• RNA modifications can regulate gene expression levels by affecting the processes of degradation, splicing, translocation and translation of various types of RNAs. For example, YTHDF family proteins are m6A-modified reading proteins that recognise and bind mRNAs modified by m6A, thereby regulating mRNA expression levels. In S. methanogenes, some mRNAs have m1A modifications and are mainly enriched in their 3'untranslated region (3'UTR). The level of m1A modifications is negatively correlated with the translation efficiency of mRNAs, which suggests that m1A may affect gene expression by inhibiting translation(Li C et al., 2024).

Affects RNA structure and function

• RNA modifications can alter the secondary and tertiary structure of modified RNAs, thereby affecting the interaction of these RNAs with other molecules. For example, specific modifications on tRNAs and rRNAs ensure their effectiveness in protein synthesis. Pseudotidylated anticodons are able to read other codons efficiently, and during mitochondrial translation if some anticodons are not pseudotidylated, it is difficult to recognise the corresponding codon. It was also shown that ψ-modification enhances mRNA translation because unmodified mRNA is more likely than ψ-modified mRNA to bind and activate an RNA-dependent protein kinase (PKR), which is responsible for the phosphorylation of translation initiation factor 2-alpha (eIF-2alpha) and reduces translation efficiency(Maassen S et al., 2023).

Involvement in RNA processing

• Studies have shown that modifications such as pseudouridine (Ψ) and N6-methyladenosine (m6A) can either facilitate or inhibit RNA splicing and processing to ensure that functionally mature RNAs can be generated. e.g., the efficiency of splicing in precursor mRNAs is significantly increased by the simultaneous presence of both Ψ and m6A modifications, an effect that may be attributed to the fact that RNA modification alters the folding state of the RNA and the ability of the RNA to bind to splicing factors to achieve this effect(Zhuang H et al., 2023).

Cell signaling

• RNA modifications play an important role in cellular signalling pathways and significantly affect cellular responses to external stimuli. For example, m6A modifications have an important role in cellular stress response and metabolism. Studies have shown that an increase in m6A modifications on certain cellular RNAs can lead to an up-regulation of the expression of some antioxidant genes, enabling cells to resist oxidative damage; m6A modifications can also affect cellular metabolic pathways, helping to adapt to the cell in nutrient-deficient environments(Zhao YZhao Y et al., 2024).

Involvement in disease mechanisms

• Abnormalities in RNA modifications have been linked to the development of a variety of diseases, including cancer, neurodegenerative diseases, and metabolic diseases. Studying changes in RNA modifications can help understand disease mechanisms and may provide new therapeutic targets.For example, many studies have shown that m6A is associated with the development of cisplatin resistance in tumors, and ALKBH5 overexpression enhances the sensitivity of bladder cancer cells to cisplatin via the CK2α-mediated m6A-dependent glycolytic pathway(Zhuang H et al., 2023).

Epigenetic regulation

• RNA modifications also play a key role in epigenetics, where they can affect the function of RNAs and, in turn, the expression patterns of the genome.For example,such modulations can achieve lasting changes in gene expression without altering the DNA sequence.When the level of m6A increases, the expression of genes related to stem cell properties (e.g. Oct4 and Sox2) is enhanced, promoting self-renewal; whereas, during differentiation, dynamic changes in m6A modification result in the up-regulation of expression of genes related to differentiation and the down-regulation of expression of genes related to stem cell properties, thus helping the cells to maintain a balance between self-renewal and differentiation(Sun W et al., 2021).

Evolution and Adaptation

• Evolutionary changes in RNA modifications across species demonstrate their importance in biological adaptation and diversity. By regulating RNA stability and function, organisms are able to rapidly adapt to environmental changes.Many studies have revealed dynamic changes in m6A methylation in plants in terms of organ, age, and adversity dependence, allowing plants to increase their resistance to better adapt to their environment(Shen L et al., 2023).

As a biomarker

• Due to the specificity and dynamics of RNA modifications, they can be used as biomarkers for early diagnosis and prognostic assessment of diseases. For example, changes in peripheral blood RNA m6A modification levels and associated modifying enzymes can provide a reference for tumour diagnosis and disease course monitoring. Studies have shown that peripheral blood RNA m6A modification levels are significantly higher in patients with breast cancer than in control subjects with benign breast disease. m6A alone or in combination with existing tumour markers or the methylase METTL14/FTO has a significantly higher diagnostic value for breast cancer than the existing breast cancer markers CEA and CA153(Ge L et al., 2020).

RNA Modifications List

Table 1 Some common RNA modifications

Eight common RNA modifications

• N6-methyladenosine (m6A): methylation of adenosine at the N6 position. It is one of the most common RNA modifications in eukaryotes and is widely found in the exonic and 3'-end untranslated regions of all types of RNAs (e.g., mRNAs and non-coding RNAs). highly conserved sequences are present near the m6A modification site on mRNAs, but there are no similarly conserved sequences for the m6A modification site in rRNAs, tRNAs, and snRNAs. m6A modifications can affect the transcription, maturation, and transport of different RNA types, and play a regulatory role in a variety of cellular processes. m6A modifications can affect the transcription, maturation, and transport of different RNA types and play a regulatory role in a variety of cellular processes.
• N1-methyladenosine (m1A): N1-methyladenosine (m1A): The m1A modification occurs at the first N atom of the adenine base. m1A first appeared in non-coding RNAs (e.g., rRNAs and tRNAs), and has since been found to be widely present in prokaryotic and eukaryotic mRNAs as well. It is associated with processes such as RNA stability, stress-induced granulation, and trophoblast invasion.
• 5-Methylcytosine(m5c): 5-Methylcytosine (m5c): m5C methylation occurs at the 5th position of cytosine residues. It is found on tRNA, rRNA, mRNA, and miRNA, and is particularly abundant in eukaryotic tRNA and rRNA. m5C methylation is a dynamic and reversible form of RNA modification, and is an important form of post-transcriptional epigenetic regulation.
• 7-methylguanylate(m7G): 7-Methylguanosine (m7G): N7-methylguanosine (m7G) refers to RNA methylation of guanosine at the N7 position. m7G can be used to cap the 5' end of mature mRNAs, snRNAs and snoRNAs. The m7G methylation is abundant in the 5'UTR, coding region (CDS), and 3'UTR of mRNAs, as well as in precursor mRNA.
• Pseudouridine (Ψ): Pseudouridine (Ψ) is a natural structural analogue of uracil nucleosides. In pseudouridine, the ribose is not attached to uracil N1 but to C5 of the pyrimidine ring. ψ is one of the most abundant forms of RNA modification, present in all types of RNA in cells, and it is highly conserved across species. Since ψ is formed by sequence-specific isomerisation of uracil (U), this modification is more abundant in both tRNA and rRNA. Ψ plays an important role in RNA biosynthesis, structure, stability, and function, and is involved in the regulation of gene expression.
• N6,2'-O-Dimethyladenine(m6Am): TN6,2'-O-dimethyladenine (m6Am): m6Am refers to the m6A modification in which the 2'hydroxyl group of the ribose of the same adenine residue is also methylated, resulting in a 2'methoxy structure (2'-O-CH3). m6Am is the modification immediately following m7G at the start of the mRNA, suggesting that the modification is located exclusively in the 5' UTR. m6Am modification is closely related to mRNA unwinding and controls RNA stability by affecting unwinding. The m6Am modification is closely related to mRNA unwinding and controls RNA stability by affecting unwinding. The m6Am modification has been found to be present in 50%-80% of mammals and plays an important role in cellular life processes by affecting the efficiency of mRNA protein translation.
• N4-acetylcytidine (ac4C): N4-acetylcytidine (ac4C): N4-acetylcytidine (ac4C) is a chemical modification conserved in eukaryotes and prokaryotes, with the presence of an acetylation group at the N4 position of cytidine. Like many RNA modifications, ac4C was initially detected in tRNA and rRNA, but was later found to be abundantly present in mRNA. ac4C is widely distributed in the human transcriptome, with the majority of sites located in coding sequences (CDSs), and is capable of promoting the expression of target genes by enhancing the stability of mRNAs and translation.
• Adenosine-to-inosine editing(A-to-I): Adenine to hypoxanthine (A-to-I) RNA editing mediated by ADAR (adenosine deaminase acting on RNA) proteins is a widespread co-transcriptional and post-transcriptional modification in postnatal animals. Because I is recognized as G, A-to-I RNA editing spatiotemporally and spatially specific increases transcriptome and proteome diversity without altering the genome sequence.A-to-I editing is widespread in precursor mRNAs, mRNAs, noncoding RNAs (e.g., miRNAs, long noncoding RNAs (lncRNAs)), tRNAs, and even viral RNAs.

The chemical structure, distribution, and molecular functions of eight RNA modifications(Liu WW et al., 2024)

RNA modification mechanisms

• Methylation: RNA methylation is the event in which RNA methylation modifying enzymes add methyl groups (-CH₃) to certain nucleotides of an RNA molecule, such as N6-methyladenosine (m6A), 5-methylcytidine (m5C), and N1-methyladenosine (m1A) RNA methylation modifications are present in several types of RNAs including messenger RNAs (mRNAs), transfer RNAs (tRNAs), RNA methylation modifications are found in several types of RNAs, including messenger RNAs (mRNAs), transfer RNAs (tRNAs), ribosomal RNAs (rRNAs) and non-coding RNAs (ncRNAs).
• Pseudouridylation: Pseudouridine on RNA is a natural structural analog of regular uridine (U) in the RNA molecule, except that in its ring structure a hydrogen bond is formed between the carbon 1 and nitrogen 1 positions, which makes it chemically stable and distinct from uridine. Pseudouridylation is an important RNA modification commonly found in tRNA, rRNA and mRNA.
• Editing: RNA editing is a post-transcriptional modification process in which RNA is edited after transcription so that the modified sequence differs from the original DNA sequence. RNA editing affects the function, stability, and translational efficiency of the RNA, as well as altering the function of proteins translated from the RNA. rna editing occurs in a wide variety of organisms, especially mammals, plants, and some viruses.a-to-i editing (adenosine to inosine editing): adenosine (A) is converted to inosine (I), which is recognised as guanosine (G) during translation, catalysed by an enzyme called ADAR (Adenosine Deaminase Acting Reacting Receptor on RNA); C-to-U editing (Cytidine to Uridine Editing): cytidine (C) is converted to uridine (U), catalysed by an enzyme called APOBEC (cytidine deaminase).
• 5' capping: RNA 5' capping is an important process of post-transcriptional modification that occurs mainly in eukaryotic mRNA molecules. Capping plays an important role in RNA stability, translation and translocation. In eukaryotes, capping is usually a 7-methylguanosine (m7G) residue attached to the 5' end of the RNA by a 5'-5' triphosphate bond. However, sometimes the 2' or 7' position of the capped guanosine is methylated, resulting in a different form of capped structure.
• Polyadenylation: RNA polyadenylation is an important process of post-transcriptional modification that occurs mainly on mRNA precursors (pre-mRNAs) in eukaryotes. This process involves the addition of a string of adenylate (A) residues to the 3' end of the RNA to form a poly(A) tail.
• Phosphorylation: The phosphorylation of certain nucleotides in RNA, such as 5' phosphorylation, 3' phosphorylation, and internal phosphorylation. This process is essential for RNA stability and translation initiation.
• Acetylation: RNA acetylation is a modification that adds an acetyl group (-COCH₃) to an RNA molecule. This process mainly involves the acetylation of specific nucleotides in RNA, especially post-transcriptionally, which are present in mRNA, tRNA, and rRNA.RNA acetylation plays an important role in the cell, affecting RNA stability, function, and interactions with other molecules.

RNA modifications in humans

RNA modifications and related regulatory factors play important roles in the maintenance of normal physiological functions and the development of cancers, such as m6A methylation dysregulation is closely associated with haematological disorders, central nervous system disorders, reproductive disorders, metabolic disorders, and cancers. m7G modifications are involved in the process of various cancers including hepatocellular carcinoma, bladder cancer, and pancreatic cancer, and have been associated with drug-resistant cancers. Several studies have shown that m6A, m7G, and 2'-O-Me modifications are associated with neuropsychiatric disorders such as depression, autism, and Alzheimer's disease. One of the RNA modifications, ac4C acetylation, has been found to be enriched in cellular stress granules, and the acetylated transcripts are mainly localised in stress granules in response to oxidative stress, regulating the cellular stress response. Moreover, a number of studies have shown that proteins regulating m6A modifications are associated with CD8+ T cell function, e.g., Ythdf1-deficient mutant mice exhibit higher antigen-specific CD8+ T cell antitumour responses. In breast cancer, the demethylase ALKBH5 mediates m6A demethylation of NANOG mRNA, leading to elevated NANOG mRNA expression and increased protein synthesis, inducing a breast cancer stem cell phenotype. In AD patients, disruption of the balance of m6A modifications affects the abnormal aggregation of pathological proteins in AD patients, and m6A modifications can interfere with the clearance of aberrant proteins by affecting the expression of key factors in the proteasomal protein degradation pathway. In systemic lupus erymatosusSLE, reduced m5C levels and low expression of the m5C regulator NSUN2 were found in CD4+ T cells, and up-regulated genes for hypermethylated m5C modifications were enriched in inflammatory pathways in SLE. It was shown that the m7G modification on tRNA and its methyltransferases METTL1, WDR4, and WBSCR22 were significantly elevated in HCC, which promoted the proliferation, migration, and invasion of hepatocellular carcinoma cells, and that in HCC, METTL1 knockdown resulted in a reduction of tRNA m7G modification, a severe impairment of mRNA translation efficiency, and reduced levels of expression of the cell cycle proteins A2, EGFR, and VEGFA. EGFR and VEGFA. In conclusion, many studies have shown that various types of RNA modifications play important roles in human diseases, stress and other physiological processes, and we need to have a deeper understanding of RNA modifications.

Through in-depth study of the mechanism of RNA modification, we can reveal the dynamics of intracellular RNA changes and how aberrant RNA modification affects gene expression and cellular physiological processes, as well as understand how RNA modification relates to the pathogenesis and mechanism of various diseases. Currently, we are actively searching for ways to effectively inhibit cancer, and the publication of many research results have shown that RNA modification is closely related to cancer development and drug resistance, etc. Therefore, the exploration of these mechanisms can help us to understand the mechanism, develop new disease biomarkers, and help us to achieve early diagnosis, early detection and early treatment of cancer, as well as open up a new pathway to promote the development of personalised therapeutic solutions. The researchers have also explored these mechanisms to develop new biomarkers for cancer.

If you want to have a better understanding, you can refer to the "RNA Modifications in Humans".

RNA modifications database

RNA modifications can regulate the processes of RNA production, transport, translation and metabolism, thereby modulating RNA function and affecting a wide range of physiological and biochemical processes in organisms, including humans. The understanding of RNA modifications has progressed considerably, and several databases have been created to facilitate the search for the many types of RNA modifications to better understand the functions of RNA modifications.

These RNA modification databases bring together scattered research results into a single platform for researchers to access and compare information. By sharing data and resources, different research teams can communicate and collaborate with each other, accelerating the output of RNA modification and other research results. In these databases we can quickly find the required RNA modification information and reduce the time spent on literature search on websites. The existence of databases can also help us discover and validate new RNA modifications and promote the development of RNA biology research. Moreover, some RNA modifications are closely related to the development of diseases (e.g., cancer), and the information provided by these databases can be aggregated together to help us identify RNA modification biomarkers and therapeutic targets for diseases. In summary, the various RNA modification databases provide an important resource to facilitate our in-depth understanding of RNA modification mechanisms. They not only support basic research, but also by may provide important information for clinical research and therapeutic strategy development.

There are several commonly used RNA databases, they focus on different areas, there are large databases that introduce multiple RNA modifications such as MODOMICS, and there are also databases that only elucidate one RNA modification such as m6A, m7G, etc., such as m6A-Atlas V2.0,m7GHub V2.0.

If you want to know more about RNA databases, please refer to "RNA Modifications Database."

RNA modification sequencing

RNA modification sequencing technology allows us to study the wide variety of chemical modifications that occur on the RNA molecule. Through high-throughput sequencing of different RNA modifications, we can find out how RNA modifications are distributed in different cells and even on different RNAs, and draw a blueprint of RNA modification distribution, as well as study the dynamics of RNA modifications and the relationship between RNA modifications and gene expression, which can help us to unravel the complex RNA regulatory network in the life process. Sequencing technology plays an important role in basic biological research, exploring the pathogenesis of diseases or developing new cancer therapies.

Chemical-assisted sequencingtechnologies

• The method extensively distinguishes modified and unmodified nucleotides in three ways: installation of biotin tags to enrich modified transcripts; base-pairing characterization to induce misincorporation or truncation in reverse transcription; and chemically induced cleavage followed by specific splice junctions. Including bsRNA-seq, ac4c-seq,Ψ-Seq、Pseudo-seq and PSIseq, and others. Although this technique can effectively distinguish between modified and unmodified nucleotides, thus improving the detection sensitivity of low abundance RNA and low stoichiometric modifications, it can only be applied to specific modifications, and cannot achieve single-base resolution, transcript level modification identification.

Antibody-based sequencing technologies

• Antibody-based strategies have been widely used to map the transcriptome of a variety of RNA modifications, including m6A/m6Am, m1A, hm5C, ac4C, and m7G, e.g., MeRIP-seq,acrip-seq. In this strategy, isolated RNA is first fragmented to 100-200 nucleotides (nt), followed by immunoprecipitation using specific antibodies to enrich for specific modified RNA fragments. The enriched RNA will be subjected to high-throughput sequencing combined with bioinformatics analysis to identify modifications of interest. This powerful enrichment capability makes the method extremely sensitive in detecting modifications in mRNAs and other low abundance rare RNA species. However, there is some error in accuracy and non-specific binding.

Enzyme/Protein-Assisted Sequencing

• Enzyme/Protein-Assisted Sequencing Techniques In addition to antibody immunoprecipitation, specific enzymes or proteins associated with RNA modifications can be used to affinity capture or edit transcripts containing modifications. This technique efficiently enriches and characterizes RNA molecules containing specific modifications by exploiting the catalytic activity of specific enzymes or the binding properties of specific proteins. Examples include m6A-REF-seq and MAZTER-seq. However, this may lead to false positive or false negative results due to limitations in antibody specificity and sensitivity.

RNA modifications nanopore sequencing

• Nanopore sequencing is a relatively new technology that enables direct, real-time sequencing of RNA without the need for additional reverse transcription and PCR amplification, thus reducing the bias that these steps can introduce. This method utilizes nanopores, which are small holes through which nucleic acid strands pass. As these strands pass through the nanopore, they cause changes in ionic currents, which can be measured and interpreted to determine the order of the nucleotides. Mechanistically, single-stranded RNA is driven through the nanopore by motor proteins, resulting in changes in ionic currents generated by a set of k-nucleotides (kmer, typically k of 5) located within the pore, which allows us to computationally decode the nucleotide sequence. In addition to sequence information, the chemical modification and secondary structure of the RNA also affects its movement through the nanopore, which in turn can be directly analyzed by computational algorithms. In addition, nanopore sequencing produces reads long enough to cover the full length of the transcript, which enables accurate identification of highly repetitive regions, splicing products, and the length of polyadenylation tails. Nanopore direct RNA sequencing promises to simultaneously identify different modifications in a single molecule, but its accuracy and sensitivity are still limited and simultaneous detection of multiple modifications is not yet possible. Therefore, further efforts are still needed to improve the instrumentation to reduce signal noise and to develop more robust computational analysis methods for the simultaneous identification of multiple modifications.

If you want to learn more about these techniques, check out the "RNA Modification Sequencing".

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