ac4C-seq vs. Other Sequencing Technologies: An Overview

As the core research object of epigenetics, RNA modification is the key mechanism for cells to regulate gene expression at the RNA level. Since pseudouracil (ψ) was first discovered in tRNA in the 1950s, more than 170 kinds of RNA chemical modifications have been identified, which participate in almost all physiological processes by changing the structural stability, intermolecular interaction, and metabolic fate of RNA. From N6-methyladenosine (m6A) and N4-acetylcytidine (ac4C) of mRNA to 5-methylcytosine (m5C) of tRNA, each modification has its unique temporal and spatial distribution and functional division:

• m6A influences stem cell differentiation by regulating mRNA splicing and degradation
• ac4C participates in stress response by enhancing translation efficiency
• m5C plays a role in RNA nuclear output, and ψ is guaranteed by stabilizing the structure of rRNA

However, the low abundance, dynamic fluctuation, and chemical heterogeneity of RNA modification have made its detection face challenges for a long time. The early methods relying on mass spectrometry can only achieve overall quantification, but can not locate modification sites. Antibody enrichment technology (such as MeRIP-seq) promotes the mapping of m6A and other modifications, but it is limited by resolution (about 100-200 nt) and cross-reactivity.

However, the methods of chemical labeling combined with high-throughput sequencing (such as ac4C-seq, ψ-seq) break through the above bottleneck through single base resolution, but they face the problems of RNA degradation and experimental complexity. In recent years, the rise of new technologies, such as Nanopore direct RNA sequencing, has gradually realized the synchronous detection of various modifications, which opens up a new path for analyzing the cross-regulatory network between modifications and also provides a new perspective for understanding the pathogenesis of diseases and developing targeted therapy.

This article comparatively analyzes ac4C-seq with other RNA modification profiling technologies (m6A-seq, m5C-seq, Ψ-seq) in methodologies, biological insights, technical challenges, solutions, and future directions.

Introduction to RNA Modifications and Their Detection

RNA modification is the core of epigenome regulation, and more than 170 types, such as ac4C and m6A, have been found to participate in many physiological processes by affecting RNA structure and function. However, its low abundance and dynamic characteristics make accurate detection quite challenging and promote the continuous innovation of detection technology.

Overview of Main RNA Modifications

RNA modification is an important research topic in the field of Epitranscriptomics, which plays a key role in regulating gene expression, RNA stability, and protein translation. These modifications widely exist in various RNA molecules such as mRNA, tRNA, and rRNA, and participate in many physiological and pathological processes such as embryonic development, cell differentiation, immune response, tumor occurrence, and development by affecting the structure, metabolism, and function of RNA.

• ac4C (N4-acetyl cytidine) plays a key role in the stability and translation of mRNA, and regulates the synthesis of protein by influencing the structure of mRNA and the interaction with related molecules.
• m6A (N6-methyladenosine) is one of the most abundant modifications in mRNA, which is involved in many processes such as splicing and degradation of mRNA, and has a wide influence on the regulation of gene expression.
• m5C (5-methyl cytidine): It will affect the structure of RNA and the output of RNA from the nucleus to the cytoplasm, and play an important role in the transport and function of RNA.
• ψ (Pseudouracil): It can stabilize the structures of tRNA and rRNA, and affect the decoding process of mRNA, thus ensuring the accuracy of protein synthesis.

Knockdown of NAT10 decreased the expression levels of mature miRNAs (Zhang et al., 2024)

Importance of High-resolution Positioning

• In functional research, it is very important to accurately detect RNA modification. As the core content of epigenomics, RNA modification is involved in regulating the whole life cycle process from mRNA splicing, transport, localization to translation efficiency, and plays a "molecular switch" role in key physiological and pathological processes such as embryonic development, immune response, and tumorigenesis.
• Only by defining the specific position of the modification can we deeply explore its mechanism in gene expression regulation and cell physiology. Traditional biochemical detection methods, such as MeRIP, can achieve modification enrichment, but the resolution can only reach the subgenomic level, and it is impossible to distinguish the functional differences of multiple modification sites on the same transcript.
• Emerging single-base resolution sequencing technologies, such as m6A-seq and ac4C-seq, improve the detection accuracy to the nucleotide level through chemical labeling or antibody-specific recognition, which makes it possible to analyze and modify the dynamic map.

RNA modifications and their distributions on different RNA subtypes (Cui et al., 2022)

Comparative Methodologies: ac4C-seq vs. Other Epitranscriptomic Techniques

The technology of analyzing RNA modification continues to evolve, providing a variety of tools for exploring the regulation of the epigenome. ac4C-seq is unique by virtue of the single-base resolution of chemical labeling, while technologies such as m6A-seq, m5C-seq, and ψ-seq have their own principles and characteristics. Comparing the principles, advantages, and disadvantages of these methods is of great significance for the accurate selection of technology and analytical modification function.

• A. ac4C-seq
  • a) The principle of ac4C-seq is to utilize the reduction reaction mediated by NaCNBH3 to make ac4C produce specific mutation characteristics, and then determine the position of ac4C by sequencing these characteristics.
  • b) Its advantages are that it has single-nucleotide resolution, can accurately locate a single base, and has high specificity, which reduces the occurrence of false positive results.
  • c) However, this technology also has limitations, and it needs optimized chemical treatment conditions. If it is not handled properly, it may affect the accuracy of the test results.
• B. m6A-seq
  • a) Based on the principle of antibody enrichment, m6A-seq uses an anti-m6A antibody to specifically bind RNA fragments containing m6A modification, and then performs sequencing analysis.
  • b) Its advantage is that it has a wide range of applicability, can be used for a variety of sample types, and has low requirements for RNA input.
  • c) However, its resolution is relatively low, about 100-200nt, and there is antibody deviation, which may lead to some modification sites being misjudged or omitted.

Molecular mechanisms underlying the regulation of mRNA stability through diverse RNA modifications (Boo et al., 2020)

• C. m5C-seq
  • a) M5C-seq is mainly based on the principle of bisulfite conversion. Under bisulfite treatment, unmodified cytosine (C) will be converted into uracil (U), while m5C remains unchanged. The location of m5C can be determined by sequencing.
  • b) This technique has the advantage of single-base resolution.
  • c) However, there is a risk of RNA degradation, and bisulfite treatment may damage the RNA structure, and it is easy to produce high false positive results.
• D. Ψ-seq
  • a) ψ-seq uses the chemical labeling method of CMCT to make pseudouracil labeled, which leads to the termination of reverse transcription in the process of reverse transcription, and locates ψ by detecting the termination signal.
  • b) Its advantage is that it can quantitatively locate ψ.
  • c) However, the signal-to-noise ratio of this technology is relatively low, which may affect the accuracy of detection.

Comparison between ac4C-seq and other epitranscriptomic techniques

Biological Insights from Comparative Studies

Different RNA modifications do not exist in isolation, and their functions are intertwined to form a complex regulatory network. Through the comparative study of ac4C-seq and m6A-seq, the functional overlap and specificity between modifications can be revealed.

Overlap and Difference of Functions

• A. ac4C and m6A
  • a) ac4C and m6A, as highly conserved RNA modifications in eukaryotes, can regulate gene expression by stabilizing mRNA structure, but there are significant differences in modification sites and functional mechanisms. ac4C modification is mainly enriched in the coding region (CDS) of mRNA, which directly promotes the translation extension efficiency by enhancing the stability of codon-anticodon base pairing.
  • b) On the other hand, the m6A modification shows a region-specific distribution, and it is highly enriched near the 3' untranslated region (3'UTR), 5'UTR, and stop codon. By recruiting YTHDF family reader proteins, it participates in the process of alternative splicing, nuclear output, translation efficiency regulation, and degradation of mRNA. In addition, the modification of m6A is closely related to physiological processes such as cell differentiation and stress response, and ac4C plays a more prominent role in regulating cell metabolism and protein synthesis.
• B. ac4C and m5C
  • a) m5C modification is widely distributed in non-coding RNA, such as tRNA and rRNA, which is mainly involved in maintaining the stability of RNA secondary structure and translation accuracy. In sharp contrast, ac4C modification takes mRNA as the core target, which directly affects the dynamic changes of protein groups by regulating the translation efficiency, half-life, and subcellular localization of mRNA.
  • b) In mammalian cells, the NSUN family of m5C modifying enzymes mainly locates in the nucleus, while the ACTR2/3 complex, the key regulatory enzyme of ac4C, mainly plays a role in the cytoplasm. This subcellular localization difference further determines the division of labor between the two modifications in the RNA metabolic pathway.

Detection of modified bases in mRNA (Kumar et al., 2021)

• C. Co-occurrence and cross-regulation
• a) The existing research evidence shows that there is a phenomenon of "modification cross-regulation". This regulatory mechanism is ubiquitous in organisms, which finely regulates gene expression through the interaction of various RNA modifications, thus affecting the physiological and pathological processes of organisms. In the stress response, m6A and ac4C will be co-enriched and participate in the regulation process of the cell coping with stress.
• b) m6A, as the most common internal modification on eukaryotic mRNA, can affect the stability, translation efficiency, and splicing process of mRNA. As a new type of RNA acetylation modification, ac4C can enhance the stability and translation ability of mRNA. Both of them affect the expression of related genes through synergy and help cells adapt to an adverse environment.
• c) Specifically, under the condition of oxidative stress, m6A modification can promote the degradation of related antioxidant gene mRNA and avoid the metabolic burden caused by over-expression. At the same time, ac4C modification can stabilize the mRNA encoding antioxidant enzymes and ensure its continuous translation to produce enough antioxidant proteins. This double-modified dynamic balance mechanism enables cells to quickly adjust their metabolism and defense strategies under stress and maintain the homeostasis of the intracellular environment.
• D. Significance of disease
• a) The role of ac4C in cancer: ac4C and m6A overlap in the regulation of oncogenes, for example, in the regulation of the MYC gene, they may jointly affect the expression of oncogenes through different mechanisms and promote the occurrence and development of cancer.
• b) Role of ψ in ribosomal diseases: ψ plays a different role in ribosomal diseases from that of ac4C in translation. ψ mainly influences the translation process by stabilizing the ribosome structure, while ac4C regulates protein synthesis by influencing the stability and translation efficiency of mRNA.

The ac4C-dependent stress granule transcriptome (Kudrin et al., 2024)

Technical Challenges and Emerging Solutions

Although RNA modification detection technology has promoted the development of epigenetics, the existing methods have their limitations: the antibody method is easy to cross-react, and the chemical method has the risk of RNA damage. These challenges restrict the study of multi-modification synergy mechanisms, and emerging technologies such as direct RNA sequencing, mass spectrometry, and machine learning are providing new paths for breaking through bottlenecks and achieving accurate and efficient detection.

Limitations of Current Methods

Antibody-based methods (such as acRIP and MeRIP) have cross-reaction and resolution problems. Antibodies may bind nonspecifically with other similar modifications, leading to cross-reaction, and it is difficult to accurately locate the modification site at low resolution.

Chemical-based methods (such as ac4C-seq, ψ-seq) have the problems of RNA damage and complicated experimental procedures. The chemical treatment process may damage RNA and affect its structure and function, and the complex experimental process increases the difficulty of operation and the possibility of experimental errors.

Innovation of Multi-modified Spectrum Analysis

• Direct RNA sequencing: This technology can detect multiple modifications at the same time, without antibody enrichment or chemical treatment, which makes it possible for simultaneous analysis of multiple modifications and greatly improves the analysis efficiency.
• Mass spectrometry: It can verify the stoichiometric ratio of modification and clarify the proportion of modification in RNA molecules, which provides important data for further understanding the functional significance of modification.
• Machine learning tool: It can predict modification sites according to the sequence background, and establish a prediction model by learning from a large number of data to help researchers find potential modification sites more quickly and accurately.

Future direction of RNA Modification Research

• Integrating ac4C-seq, m6A-seq, and m5C-seq data sets to construct a unified epigenome map will help to fully understand the relationship between different RNA modifications and their overall role in gene expression regulation, and provide a more comprehensive perspective for in-depth study of the molecular mechanism of life activities.
• The method of combining single cell RNA sequencing (scRNA-seq) with modification spectrum analysis, from the existing scm6A-seq to the future scac4C-seq, can study the heterogeneity of RNA modification at the single cell level and reveal the differences of RNA modification in different cell types and their relationship with cell function.

EV2,Enrichment innormaПzecexpression and ac4C counts on transcripts (Kudrin et al., 2024)

Conclusion

ac4C-seq, m6A-seq, m5C-seq, ψ-seq, and other technologies together form a multidimensional tool matrix for analyzing RNA modification. Although there are differences in principle and performance among these technologies, ac4C-seq is good at single-base resolution and mRNA targeting, m6A-seq is better at universality, and m5C-seq and ψ-seq focus on specific modification types, respectively, but their cooperative application is revealing the complex relationship between modifications.

In the future, with the maturity of nano-hole direct sequencing and other technologies, simultaneous detection of multiple modifications will break through the existing limitations and promote the construction of a unified apparent transcriptome map. The analysis of modified spectra at the single-cell level and the research and development of drugs targeting the "writing enzyme" will further deepen our understanding of disease mechanisms and provide a new paradigm for precise treatment. The integration and innovation of these technologies will eventually reveal the whole picture of the RNA modification network regulating life activities.

References

1. Zhang H, Lu R, Huang J, et al. "N4-acetylcytidine modifies primary microRNAs for processing in cancer cells." Cell Mol Life Sci. 2024 81(1): 73.
2. Cui L, Ma R, Cai J, et al. "RNA modifications: importance in immune cell biology and related diseases." Signal Transduct Target Ther. 2022 7(1): 334.
3. Boo, S.H., Kim, Y.K. "The emerging role of RNA modifications in the regulation of mRNA stability." Exp Mol Med. 2020 52: 400-408.
4. Kumar S, Mohapatra T. "Deciphering Epitranscriptome: Modification of mRNA Bases Provides a New Perspective for Post-transcriptional Regulation of Gene Expression." Front Cell Dev Biol. 2021 9: 628415.
5. Kudrin P, Singh A, Meierhofer D, Kuśnierczyk A, Ørom UAV. "N4-acetylcytidine (ac4C) promotes mRNA localization to stress granules." EMBO Rep. 2024 25(4):1814-1834.