Fundamentals of ac4C-seq: Principles Applications and Beyond

RNA modification refers to the process of chemical modification of nucleotide residues through a series of enzymatic reactions after RNA molecules are synthesized. These modifications are widely found in various RNA molecules, such as mRNA, tRNA, rRNA, etc., which add extra regulatory levels to the structure and function of RNA, and are called "epigenome". At present, more than 170 different types of RNA modifications have been found, which play a key role in RNA stability, translation efficiency, localization, and interaction with other molecules. As a relatively newly discovered RNA modification, N4-acetyl cytidine (ac4C) is gradually becoming a research hotspot. Understanding the modification of ac4C RNA is of great significance to reveal the complex regulation mechanism of RNA and the pathogenesis of related diseases.

The article comprehensively introduces the fundamentals of ac4C-seq, bioinformatics pipelines, and biological insights from related studies, as well as concluding with existing problems and future prospects.

Introduction to ac4C RNA Modification

ac4C is a key chemical modification of RNA, and acetyl groups are added by acetyltransferase catalysis. As an important member of the regulation of epigenome, it is widely involved in the maintenance of mRNA stability and the regulation of translation efficiency, and plays a significant role in physiological and pathological processes such as cell stress and tumorigenesis, which provides a new perspective for the analysis of RNA function regulation network.

ac4C and mRNA Stability

Studies have shown that ac4C modification is closely related to the stability of mRNA. Acetyltransferase NAT10 can catalyze the formation of ac4C modification on mRNA. When NAT10 is deleted by gene knockout, the detection amount of ac4C at the located mRNA site is significantly reduced, and it is related to the down-regulation of the target mRNA as a whole.

The analysis of the half-life of mRNA shows that the stability of mRNA with acetylation modification is dependent on the increase of NAT10. This indicates that ac4C modification can enhance the stability of mRNA and reduce its degradation rate, thus prolonging the existence time of mRNA in cells and providing a more stable template for the subsequent translation process.

ac4C and mRNA Translation

ac4C modification also plays an important role in mRNA translation. In vitro and in vivo experiments have confirmed that acetylation of mRNA can enhance the translation efficiency of substrates. Through the analysis of codon content in the ac4C peak, it is found that there is a cytidine bias at the wobble site, and this bias will affect the decoding efficiency of mRNA. Specifically, ac4C modification may promote the binding and movement of ribosomes on mRNA by changing the interaction between mRNA and translation-related molecules such as ribosomes and translation initiation factors, thus improving the accuracy and efficiency of translation and enabling cells to synthesize protein more effectively.

Localization sites of ac4C modification in mRNA, tRNA, and rRNA (Zhang et al., 2024)

Comparison with Other RNA Modifications

• A. m6A modification
  • a) N6-methyladenosine (m6A) is one of the most widely studied RNA modifications. M6A modification mainly occurs in RRACH (R=G or a; H=A, c or u); its modification sites are widely distributed in the transcription group. Similar to ac4C, m6A modification is also involved in the regulation of mRNA stability, translation, and splicing. However, there are differences between them in modification site preference, regulatory mechanism, and functional influence.
  • b) m6A modifications are mostly concentrated near the 3' untranslated region (3'UTR) and stop codon of mRNA, while ac4C modifications are more abundant in the coding sequence.
  • c) In terms of regulatory mechanisms, m6A modification mainly recognizes and mediates its function through specific code readers (such as YTHDF family proteins), while ac4C modification may play a role by affecting the secondary structure of mRNA or directly interacting with translation-related factors.
  • d) In terms of function, m6A modification plays an important role in embryo development and cell differentiation, while ac4C modification has unique advantages in maintaining mRNA stability and promoting translation efficiency.
• B. ψ modification
  • a) Pseudouracil (ψ) modification is a modification that changes the glycosidic bond of uracil from the N1 position to the C5 position, which is widely found in various RNA molecules. ψ modification mainly affects the secondary and tertiary structure of RNA, and enhances the stability and resistance of RNA to nuclease. Compared with ac4C, ψ-modified sites are more diverse and can occur almost anywhere in RNA.
  • b) Functionally, ψ modification is very important to maintain the structure and function of tRNA and rRNA, and participates in the decoding accuracy and ribosome function in the process of protein synthesis; However, ac4C mainly regulates stability and translation at the mRNA level.
  • c) In addition, their modifying enzymes and recognition mechanisms are completely different; ψ modification is catalyzed by the pseudouracil synthetase family, while ac4C is catalyzed by NAT10.

NAT10-related mechanisms involved in tumor progression (Zhang et al., 2024)

Development of ac4C Sequencing Technologies

As a key RNA modification, ac4C's functional analysis depends on the breakthrough of sequencing technology. It is difficult to locate the modification site accurately by relying on low-resolution detection methods in the early stage. With the integration of molecular biology and sequencing technology, ac4C sequencing has been gradually upgraded from acRIP-seq with antibody enrichment to a single-base resolution method, which has promoted the drawing of modified maps at the level of the complete transcriptome and laid a technical foundation for exploring its biological functions.

• A. Chemical Labeling and Antibody Enrichment
  • a) Early detection of ac4C mainly depends on chemical labeling and antibody enrichment. The chemical labeling method is to chemically treat RNA to make an ac4C site with a specific label, and then use affinity capture and other technologies to enrich the labeled RNA fragments for analysis.
  • b) The strategy based on antibody enrichment is to separate the RNA fragments containing ac4C modification from the complex RNA mixture by immunoprecipitation with antibodies that specifically recognize ac4C, and then sequence the enriched RNA by combining with high-throughput sequencing technology (such as RNA-seq), to locate the ac4C modification site at the transcriptome level.
  • c) However, these early methods have some limitations, such as chemical labeling may damage the RNA structure, and the specificity and affinity of antibodies may also affect the enrichment effect, resulting in low accuracy and resolution of detection.
• B. Evolution from acRIP-seq to High-resolution ac4C-seq
  • a) With the development of technology, ac4C RNA immunoprecipitation sequencing (acRIP-seq) technology appeared, which can detect ac4C modification sites in the whole transcription group based on an antibody enrichment strategy. However, the resolution of acRIP-seq is relatively low, so it is difficult to accurately determine the specific nucleotide position modified by ac4C. In order to improve the resolution, the researchers further developed the high-resolution ac4C-seq technology.
  • b) The detection of ac4C modification sites with single-nucleotide resolution can be realized by using a chemical-assisted method combined with deep sequencing. By special chemical treatment of RNA, these technologies make ac4C modification sites generate unique signals in the sequencing process, thus accurately identifying the nucleotide sequence of modification sites, greatly improving the accuracy and precision of detection of ac4C modification sites, and providing a powerful tool for in-depth study of the function and mechanism of ac4C modification.

Pri-miRNAs are ac4C-modified (Zhang et al., 2024)

Bioinformatics Pipelines for ac4C Detection

As a key RNA modification, ac4C genome-wide localization depends on a bioinformatics analysis process. This process connects sequencing data with biological interpretation, and through data preprocessing, peak identification, modification site annotation, and other steps, the distribution law and functional characteristics of ac4C are mined from sea sequencing information. Accurate bioinformatics analysis is the core link to analyze the regulation mechanism of ac4C, and also lays the foundation for the study of related diseases.

Peak Identification Tool

In the analysis of ac4C-seq data, peak identification is one of the key steps to determine the enrichment region of ac4C modification on the genome. Commonly used peak identification tools include MacS2 (model-based analysis of chip-seq data 2) and exomePeak2.

• MACS2 evaluates the enrichment of reads in sequencing data by establishing a Poisson distribution model, and can effectively identify statistically significant ac4C enrichment peaks. It takes into account the background noise of sequencing data and accurately locates the ac4C modification site by comparing the sample with the control data.
• ExomePeak2 is a peak identification tool specifically for exon sequencing data. When processing ac4C-seq data, it can accurately identify the enriched peaks by its unique algorithm, and at the same time, it can analyze the information such as the intensity and position of the peaks in detail, which provides an important basis for subsequent functional annotation and analysis.

Distinguish ac4C from Other Modifications.

Due to the diversity and complexity of RNA modifications, it presents a significant challenge to distinguish ac4C from other modifications in data analysis. Different RNA modifications may coexist on the same RNA molecule, and their modification sites may overlap. For example, both m6A modification and ac4C modification are distributed in mRNA, and some sites may be affected by both alterations at the same time.

In addition, some modified chemical structures are similar, and the signals generated during sequencing may be confused, which makes it difficult to distinguish them accurately. To solve this problem, it is necessary to comprehensively use a variety of analytical methods, such as immunoprecipitation sequencing by combining specific antibodies with different modifications, comparing the distribution characteristics of different modifications on the genome, and deep mining and analysis of sequencing data by bioinformatics algorithm, to improve the accuracy of identifying ac4C modifications through multi-dimensional information integration.

NAT10 is highly expressed in cancers and negatively correlated with poor prognosis (Zhang et al., 2024)

Biological Insights from ac4C-seq Studies

The development of ac4C-seq technology provides a powerful tool for analyzing the biological function of ac4C modification. By locating the ac4C modification site at the whole transcriptome level, researchers gradually revealed the key role of this modification in the process of cell physiology and pathology, and its close relationship with the occurrence and development of diseases and cell stress response is becoming an important breakthrough in the study of epigenomics.

Association with Disease

• A. Cancer
  • a) More and more studies show that ac4C modification is closely related to the occurrence and development of cancer. In many cancers, such as esophageal squamous cell carcinoma, the modification of ac4C mediated by NAT10 leads to the stability and overexpression of long-chain noncoding RNA CTC-490G23.2, which is further up-regulated in primary esophageal squamous cell carcinoma and its metastatic tissues.
  • b) In addition, the abnormal expression of genes related to ac4C modification was also found in other cancer types, indicating that ac4C modification may play an important role in the occurrence, development, metastasis, and prognosis of cancer, and it is expected to become a new target for cancer diagnosis and treatment.
• B. Virus infection
  • a) In the process of virus infection, ac4C modification is also involved. After infecting host cells, some viruses will use the RNA modification mechanism of host cells to regulate the expression of their genes and the life cycle of the virus. For example, the mRNA of some viruses may be modified by ac4C, which may affect the stability and translation efficiency of viral mRNA, thus affecting the replication and spread of viruses.
  • b) At the same time, the host cell may also respond to virus infection and initiate an immune response by adjusting its ac4C modification level. An in-depth study on the role of ac4C modification in viral infection will help reveal the molecular mechanism of viral infection and provide new ideas for developing antiviral drugs and treatment strategies.

Functional Significance in Cell Stress Response

In cell stress response, ac4C modification plays a key role in dynamic regulation. When cells face environmental stress such as oxidative stress, nutritional deficiency, or high temperature, the modification of ac4C mediated by NAT10 will selectively enrich the mRNA of stress-related genes, and quickly start the stress defense pathway by enhancing its stability and improving translation efficiency.

Under heat shock conditions, ac4C modification can target heat shock protein (HSP) family mRNA and accelerate its translation to protect cells from protein denaturation. However, during nutrient deprivation, ac4C maintains the basic energy supply of cells by stabilizing metabolism-related mRNA. This modified stress-specific regulation provides an efficient apparent transcription regulation mechanism for cells to quickly adjust gene expression profiles and maintain survival stability in an adverse environment.

ldentification of the NAT10 targets using RNA sequencing (Dang et al., 2024)

Conclusion

To sum up, ac4C RNA modification, as an important epigenome modification, plays a unique and key role in mRNA stability and translation regulation. Compared with other RNA modifications, ac4C modification has its own characteristics and functional advantages. From the biological point of view, ac4C modification is closely related to the occurrence and development of many diseases (such as cancer and virus infection), and has important functional significance in cell stress response.

However, the research on ac4C modification is still at a relatively early stage, and there are still many problems to be solved urgently. Although NAT10 is known as the key enzyme to catalyze ac4C modification, its regulatory mechanism under different cell types and physiological and pathological conditions is not completely clear. The ac4C-modified code readers and their functions of recognizing ac4C-modified mRNA and how to precisely regulate it need to be further identified and studied.

With the deepening of research, it is believed that ac4C RNA modification will provide us with a new perspective and strategy to understand the complexity of life processes and overcome related diseases.

References

1. Zhang S, Liu Y, Ma X, et al. "Recent advances in the potential role of RNA N4-acetylcytidine in cancer progression." Cell Commun Signal. 2024 22(1): 49.
2. 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.
3. Dang Y, Li J, Li Y, et al. "N-acetyltransferase 10 regulates alphavirus replication via N4-acetylcytidine (ac4C) modification of the lymphocyte antigen six family member E (LY6E) mRNA." J Virol. 2024 98(1): e0135023.