Quantifying m⁶A and Ψ Modifications in the Transcriptome via Chemical-Assisted Approaches

Since the discovery of the first chemically modified RNA nucleotide in 1951, more than 170 types of chemical modifications have been characterized in RNA so far. Since the discovery of the reversible and dynamic nature of N⁶-methyladenosine (m⁶A) in mRNA modification, researchers have identified about ten modifications in eukaryotic mRNA; together with modifications on the noncoding RNAs, the term "epitranscriptome" has been coined to describe the ensemble of various chemical RNA modifications. The past decade has witnessed the discovery of many novel molecular functions of mRNA modifications, demonstrating their crucial roles in gene expression regulation. As the most abundant modifications in mRNA, the study of m⁶A and Ψ has been facilitated by innovative high-throughput sequencing technologies, which can be based on antibodies, enzymes, or novel chemistry. Among them, chemical-assisted methods utilize selective chemistry that can discriminate modified versus unmodified nucleotides, enabling the transcriptome-wide mapping of m⁶A and Ψ modifications and functional studies.Our group has developed several sequencing technologies to investigate these epitranscriptomic marks including m⁶A, Ψ, m¹A, and m⁶Am. Among them, we have recently developed two methods for absolute quantification of m⁶A and Ψ in the transcriptome based on chemical reactivity to distinguish and measure the two modifications. In GLORI, we used glyoxal and nitrite to mediate efficient deamination of regular adenosine, while m⁶A remained unaffected, thereby enabling efficient and unbiased detection of single-base resolution and absolute quantification of m⁶A modification. In CeU-seq and PRAISE, we used different chemistry to achieve selective labeling and detection of transcriptome-wide Ψ. CeU-seq is based on an azido-derivatized carbodiimide compound, while PRAISE utilizes the unique activity of bisulfite to Ψ. PRAISE results in the formation of ring-opening Ψ-bisulfite adduct and quantitatively detects Ψ as 1-2 nt deletion signatures during sequencing. The resulting base-resolution and stoichiometric information expanded our understanding to the profiles of RNA modifications in the transcriptome. In particular, the quantitative information on RNA methylome is critical for characterizing the dynamic and reversible nature of RNA modifications, for instance, under environmental stress or during development. Additionally, base-resolution and stoichiometric information can greatly facilitate the analysis and characterization of functional modification sites that are important for gene expression regulation, especially when one modification type may have multiple or even opposing functions within a specific transcript. Together, the quantitative profiling methods provide the modification stoichiometry information, which is critical to study the regulatory roles of RNA modifications.In this Account, we will focus on the quantitative sequencing technologies of m⁶A and Ψ developed in our group, review recent advances in chemical-assisted reactions for m⁶A and Ψ detection, and discuss the challenges and future opportunities of transcriptome-wide mapping technologies for RNA modifications.