AlkB-Facilitated Demethylation Enables Quantitative and Site-Specific Detection of Dual Methylation of Adenosine in RNA

Abstract

RNA molecules undergo various chemical modifications that play critical roles in a wide range of biological processes. N 6 ,N 6 -Dimethyladenosine (m 6,6 A) is a conserved RNA modification and is essential for the processing of rRNA. To gain a deeper understanding of the functions of m 6,6 A, site-specific and accurate quantification of this modification in RNA is indispensable. In this study, we developed an AlkB-facilitated demethylation (AD-m 6,6 A) method for the site-specific detection and quantification of m 6,6 A in RNA. The N 6 ,N 6 -dimethyl groups in m 6,6 A can cause reverse transcription to stall at the m 6,6 A site, resulting in truncated cDNA. However, we found that Escherichia coli AlkB demethylase can effectively demethylate m 6,6 A in RNA, generating full-length cDNA from AlkB-treated RNA. By quantifying the amount of full-length cDNA produced using quantitative real-time PCR, we were able to achieve site-specific detection and quantification of m 6,6 A in RNA. Using the AD-m 6,6 A method, we successfully detected and quantified m 6,6 A at position 1851 of 18S rRNA and position 937 of mitochondrial 12S rRNA in human cells. Additionally, we found that the level of m 6,6 A at position 1007 of mitochondrial 12S rRNA was significantly reduced in lung tissues from sleep-deprived mice compared with control mice. Overall, the AD-m 6,6 A method provides a valuable tool for easy, accurate, quantitative, and site-specific detection of m 6,6 A in RNA, which can aid in uncovering the functions of m 6,6 A in human diseases.

Simultaneous Detection of Adenosine-to-Inosine Editing and N6-Methyladenosine at Identical RNA Sites through Deamination-Assisted Reverse Transcription Stalling

“…Beyond the four fundamental nucleobases of adenine, cytosine, guanine, and uracil that constitute the genetic code, RNA molecules are subject to a wide variety of chemical modifications. , These modifications have been identified in approximately 160 different forms across various RNA species spanning the biological three-domain system. − This diversity of modifications underscores the complexity and adaptability of RNA function beyond its canonical roles in protein synthesis and gene regulation . Similar to the way DNA modifications influence gene expression and genomic stability, modifications in RNA play crucial roles in fine-tuning a multitude of biological processes, including the regulation of mRNA stability and translation, RNA splicing, transport, and localization. − …”

Selective Ligase-Based Sample Processing-Free Discrimination and Detection of Site-Specific DNA 5-Hydroxymethylcytosine

“…Identification and detection of DNA epigenetic modifications both in vitro and in vivo are of great significance for revealing their biological functions and mechanisms of action. − Among the various epigenetic modifications, 5-hydroxymethylcytosine (5hmC) is considered as the sixth base in the genome of higher organisms, − which is of utmost significance for gene regulation during cell development and carcinogenesis . Aberrant levels of 5hmC will lead to occurrence of many diseases, including breast cancer, papillary thyroid carcinoma, and medulloblastoma, and thus the 5hmC level is a potential indicator for cancer diagnosis. − Up to now, however, some functions of 5hmC and its role in diseases are still unclear.…”