Essential Discoveries and Tools in Epitranscriptomics

The field of epigenetics has traditionally concentrated more on DNA and how changes like methylation and phosphorylation of histones impact gene expression and regulation. However, our increased understanding of RNA modifications and their importance in cellular processes has led to a rise in epitranscriptomics research. “Epitranscriptomics brings together the concepts of epigenetics and gene expression,” explained Adrien Leger, PhD, Principal Research Scientist on Modified Bases at Oxford Nanopore. “It refers to the universe of RNA modifications that influence the structure, molecular interactions, and other characteristics of transcripts.” Leger noted that while the changes don’t alter the sequence of the RNA, they have a big impact on its function.

Liz Tseng, Associate Director of Product Marketing and RNA and Gene Therapy Applications at Pacific Biosciences, emphasized how these post-transcriptional modifications can affect RNA splicing, stability, and translation, which fundamentally affect the function of a cell. “There are potentially over 170 RNA modifications and only a handful of them have been studied,” Tseng added. “Understanding and cataloging the full breadth of RNA modifications will not only increase our understanding of basic biology but will likely uncover one of the final links from genetic variation to disease.”

Among these various modifications, N⁶-methyladenosine (m⁶A) is the most well-studied, as well as the most abundant modification for mammalian mRNA¹. This modification alone has been linked to gene regulation², RNA stability³, cancer⁴, and more. Some of the other prominent modifications include N¹-methyladenosine (m¹A), 7-methylguanosine (m⁷G), pseudouridine (Ψ), and 5-methylcytidine (m⁵C)⁵. These modifications can be found in different types of RNAs including messenger RNA (mRNA), ribosomal RNA (rRNA), transfer RNA (tRNA), and long noncoding RNA (lncRNA). As researchers continue to investigate the roles of these modifications, it becomes increasingly clear that they are fundamental to many cellular processes.

How is Epitranscriptomics Studied?
One popular approach to studying epitranscriptomics involves mass spectrometry⁶. The analytical precision and sensitivity of mass spectrometry enable the identification and quantification of various modifications, but it has some limitations due to coverage depth and fragment length⁷. Over the years, many sequencing-based approaches have been developed to address the complexity of the epitranscriptome. In particular, MeRIP-seq (methylated RNA immunoprecipitation sequencing) is a popular method that employs antibodies to specifically target and immunoprecipitate RNA fragments with the m⁶A modification^8,9. This technique enables the identification of methylated regions, which are revealed as peaks in the coverage of transcripts derived from immunoprecipitated RNA relative to the input RNA.

DART-seq (deamination adjacent to RNA modification targets), is another widely used method that stands out for being an antibody-free approach for detecting m⁶A. Using a fusion protein that combines cytosine deaminase APOBEC1 with an m⁶A-binding YTH domain, this method induces a C to U deamination adjacent to m⁶A sites, which is identified using standard RNA sequencing¹⁰. Kate Myer’s lab at Duke University, the creators of DART-seq, expanded on this premise to facilitate single-cell resolution of m⁶A profiling¹¹. Additionally, the Meyer Lab recently presented GEMS (Genetically Encoded m⁶A Sensor), a novel system that signals the presence of m⁶A on mRNA through a change in fluorescence intensity. Instead of a sequencing-based approach, this technique allows for the real-time observation of m⁶A levels in live cells and tissues¹².

A significant advancement in the field of epitranscriptomics was the introduction of long-read sequencing technologies¹³. These platforms have overcome many of the challenges presented by short-read instruments and can provide deeper insights into the epitranscriptome. The current long-read sequencing approaches include single-molecule real-time (SMRT) sequencing by Pacific Biosciences (PacBio) and nanopore-based sequencing by Oxford Nanopore Technologies (ONT).

Leger emphasized how ONT’s molecular sensing tools have been particularly impactful due to their ability to directly analyze native RNA molecules. “By avoiding the conversion of RNA to cDNA, which strips away valuable epigenetic data, we give scientists a more complete and accurate view of RNA biology,” he stated. ONT’s RNA sequencing chemistry allows the detection of both the nucleic acid sequence and the RNA modifications acquired in real time. “Importantly, the technology also makes it possible to sequence full-length transcripts and estimate polyA length,” Leger explained.

In addition, Tseng shared how PacBio long-read sequencing is uniquely suited for studying RNA modifications because it can sequence the entire transcript isoform. “This means if there are multiple modifications on the same isoform, it can be reliably detected and linked.” Tseng also noted how many of the epitranscriptomics methods previously developed for short reads can be easily applied to long-read sequencing, as they use the same chemical modification or antibody-based detection. While some of the previous barriers were throughput and cost, developments in long-read sequencing have addressed these issues and established these technologies as essential tools for investigating the epitranscriptome.

Recent Discoveries
“Researchers have generated novel information related to infectious diseases, cancer, and other applications,” stated Leger. “The epitranscriptomic layer has been a key component in many of those studies, with never-before-seen data about RNA modifications powering many discoveries.” Leger then shared examples of key discoveries that highlight the importance epitranscriptome research.

One such example is a study from the onset of the pandemic that provided a detailed analysis of the SARS-CoV-2 transcriptome and epitranscriptome¹⁴. This work revealed a range of viral RNA transcripts and modifications and provided a high-resolution map showing numerous discontinuous transcription events, unknown open reading frames (ORFs), and potential RNA modification sites. The study presented the architecture of SARS-CoV-2's RNA and suggested new directions for understanding its life cycle and pathogenicity. Leger also cited another impactful study that demonstrated the application of nanopore sequencing to identify and monitor 17 different types of RNA modifications in E. coli ribosomes under varying conditions¹⁵. This approach allowed for real-time observation of ribosomal RNA changes and deepened our understanding of cellular responses and the functional implications of RNA modifications.

Tseng highlighted notable research by Liu et al. (2023), which introduced L-GIREMI, an innovative method for analyzing transcriptome data¹⁶. This approach revealed the co-occurrence of adenosine-to-inosine (A-to-I) editing sites within Alu elements. Moreover, the study showed allele-specific RNA editing events in single molecules and double-stranded RNA structures using PacBio long-read RNA sequencing data. Tseng emphasized the importance of this method’s ability to overcome the limitations of short-read technologies that struggle to sequence entire transcripts, thereby offering a more comprehensive view of RNA editing dynamics.

Another recent study investigated the mechanism behind the m⁶A modification in RNA. The researchers were able to identify DDX21 as a key factor in recruiting the m⁶A methyltransferase complex to nascent RNA, and linking R-loops, m6A modification, and transcription termination to genome stability. This discovery provided insights into the co-transcriptional regulation of m⁶A metabolism and its potential link to diseases and disorders¹⁷.

Several studies have also explored m⁶A as a prognostic signature in various cancer types. In renal cell carcinoma, the analysis of m⁶A modifications identified 9644 genes with altered m⁶A levels¹⁸. The researchers found a significant correlation between m⁶A modifications and gene expression, particularly in genes involved in renal and metabolic pathways, demonstrating potential new targets for therapy. Research focused on detecting m⁶A modifications in lncRNAs of glioblastoma cells showed 24 overlapping m⁶A-modified lncRNAs associated with glioma severity and patient survival¹⁹. Finally, a study investigating the m⁶A levels in acute myeloid leukemia (AML) patients showed a significant decrease after azacytidine plus venetoclax treatment. The researchers also identified three genes with reduced m⁶A and expression levels linked to AML prognosis, suggesting the potential of new biomarkers for AML treatment²⁰.

These studies are just a fraction of the remarkable research in epitranscriptomics. “The field of RNA biology is booming and exciting discoveries will come from areas we do not even anticipate yet,” stated Leger. He noted that he’s particularly fascinated by applications in RNA virus biology and mRNA vaccines, as well as large-scale functional transcriptomic studies. “I think we can expect scientists to ask bolder questions than ever before, and I cannot wait to see what happens when they do.”

References

1. Wang, X., Zhao, B. S., Roundtree, I. A., Lu, Z., Han, D., Ma, H., Weng, X., Chen, K., Shi, H., & He, C. (2015). N(6)-methyladenosine Modulates Messenger RNA Translation Efficiency. Cell, 161(6), 1388–1399. https://doi.org/10.1016/j.cell.2015.05.014
2. Jiang, X., Liu, B., Nie, Z., Duan, L., Xiong, Q., Jin, Z., Yang, C., & Chen, Y. (2021). The role of m6A modification in the biological functions and diseases. Signal Transduction and Targeted Therapy, 6(1), 74. https://doi.org/10.1038/s41392-020-00450-x
3. Huang, H., Weng, H., Sun, W., Qin, X., Shi, H., Wu, H., Zhao, B. S., Mesquita, A., Liu, C., Yuan, C. L., Hu, Y. C., Hüttelmaier, S., Skibbe, J. R., Su, R., Deng, X., Dong, L., Sun, M., Li, C., Nachtergaele, S., Wang, Y., … Chen, J. (2018). Recognition of RNA N⁶-methyladenosine by IGF2BP proteins enhances mRNA stability and translation. Nature Cell Biology, 20(3), 285–295. https://doi.org/10.1038/s41556-018-0045-z
4. Xue, C., Chu, Q., Zheng, Q., Jiang, S., Bao, Z., Su, Y., Lu, J., & Li, L. (2022). Role of main RNA modifications in cancer: N⁶-methyladenosine, 5-methylcytosine, and pseudouridine. Signal Transduction and Targeted Therapy, 7(1), 142. https://doi.org/10.1038/s41392-022-01003-0
5. Imbriano, C., Moresi, V., Belluti, S., Renzini, A., Cavioli, G., Maretti, E., & Molinari, S. (2023). Epitranscriptomics as a New Layer of Regulation of Gene Expression in Skeletal Muscle: Known Functions and Future Perspectives. International Journal of Molecular Sciences, 24(20), 15161. https://doi.org/10.3390/ijms242015161
6. Amalric, A., Bastide, A., Attina, A., Choquet, A., Vialaret, J., Lehmann, S., David, A., & Hirtz, C. (2022). Quantifying RNA modifications by mass spectrometry: a novel source of biomarkers in oncology. Critical Reviews in Clinical Laboratory Sciences, 59(1), 1–18. https://doi.org/10.1080/10408363.2021.1958743
7. Lauman, R., Kim, H. J., Pino, L. K., Scacchetti, A., Xie, Y., Robison, F., Sidoli, S., Bonasio, R., & Garcia, B. A. (2023). Expanding the Epitranscriptomic RNA Sequencing and Modification Mapping Mass Spectrometry Toolbox with Field Asymmetric Waveform Ion Mobility and Electrochemical Elution Liquid Chromatography. Analytical Chemistry, 95(12), 5187–5195. https://doi.org/10.1021/acs.analchem.2c04114
8. Meyer, K. D., Saletore, Y., Zumbo, P., Elemento, O., Mason, C. E., & Jaffrey, S. R. (2012). Comprehensive analysis of mRNA methylation reveals enrichment in 3' UTRs and near stop codons. Cell, 149(7), 1635–1646. https://doi.org/10.1016/j.cell.2012.05.003
9. Dominissini, D., Mo****ch-Moshkovitz, S., Schwartz, S., Salmon-Divon, M., Ungar, L., Osenberg, S., Cesarkas, K., Jacob-Hirsch, J., Amariglio, N., Kupiec, M., Sorek, R., & Rechavi, G. (2012). Topology of the human and mouse m6A RNA methylomes revealed by m6A-seq. Nature, 485(7397), 201–206. https://doi.org/10.1038/nature11112
10. Meyer K. D. (2019). DART-seq: an antibody-free method for global m⁶A detection. Nature Methods, 16(12), 1275–1280. https://doi.org/10.1038/s41592-019-0570-0
11. Tegowski, M., Flamand, M. N., & Meyer, K. D. (2022). scDART-seq reveals distinct m⁶A signatures and mRNA methylation heterogeneity in single cells. Molecular Cell, 82(4), 868–878.e10. https://doi.org/10.1016/j.molcel.2021.12.038
12. Marayati, B. F., Thompson, M. G., Holley, C. L., Horner, S. M., & Meyer, K. D. (2024). Programmable protein expression using a genetically encoded m⁶A sensor. Nature Biotechnology, 10.1038/s41587-023-01978-3. Advanced online publication. https://doi.org/10.1038/s41587-023-01978-3
13. Lucas, M. C., & Novoa, E. M. (2023). Long-read sequencing in the era of epigenomics and epitranscriptomics. Nature Methods, 20(1), 25–29. https://doi.org/10.1038/s41592-022-01724-8
14. Kim, D., Lee, J. Y., Yang, J. S., Kim, J. W., Kim, V. N., & Chang, H. (2020). The Architecture of SARS-CoV-2 Transcriptome. Cell, 181(4), 914–921.e10. https://doi.org/10.1016/j.cell.2020.04.011
15. Fleming, A. M., Bommisetti, P., Xiao, S., Bandarian, V., & Burrows, C. J. (2023). Direct Nanopore Sequencing for the 17 RNA Modification Types in 36 Locations in the E. coli Ribosome Enables Monitoring of Stress-Dependent Changes. ACS Chemical Biology, 18(10), 2211–2223. https://doi.org/10.1021/acschembio.3c00166
16. Liu, Z., Quinones-Valdez, G., Fu, T., Huang, E., Choudhury, M., Reese, F., Mortazavi, A., & Xiao, X. (2023). L-GIREMI uncovers RNA editing sites in long-read RNA-seq. Genome Biology, 24(1), 171. https://doi.org/10.1186/s13059-023-03012-w
17. Hao, J. D., Liu, Q. L., Liu, M. X., Yang, X., Wang, L. M., Su, S. Y., Xiao, W., Zhang, M. Q., Zhang, Y. C., Zhang, L., Chen, Y. S., Yang, Y. G., & Ren, J. (2024). DDX21 mediates co-transcriptional RNA m⁶A modification to promote transcription termination and genome stability. Molecular Cell, S1097-2765(24)00188-6. Advanced online publication. https://doi.org/10.1016/j.molcel.2024.03.006
18. Li, H., Li, C., Zhang, Y., Jiang, W., Zhang, F., Tang, X., Sun, G., Xu, S., Dong, X., Shou, J., Yang, Y., & Chen, M. (2024). Comprehensive analysis of m⁶ A methylome and transcriptome by Nanopore sequencing in clear cell renal carcinoma. Molecular Carcinogenesis, 63(4), 677–687. https://doi.org/10.1002/mc.23680
19. Krusnauskas, R., Stakaitis, R., Steponaitis, G., Almstrup, K., & Vaitkiene, P. (2023). Identification and comparison of m6A modifications in glioblastoma non-coding RNAs with MeRIP-seq and Nanopore dRNA-seq. Epigenetics, 18(1), 2163365. https://doi.org/10.1080/15592294.2022.2163365
20. Zhang, Z., Zhang, L., Li, J., Feng, R., Li, C., Liu, Y., Sun, G., Xiao, F., & Zhang, C. (2024). Comprehensive analysis of m⁶A methylome alterations after azacytidine plus venetoclax treatment for acute myeloid leukemia by nanopore sequencing. Computational and Structural Biotechnology Journal, 23, 1144–1153. https://doi.org/10.1016/j.csbj.2024.02.029