The precise and unambiguous detection and quantification of internal RNA modifications represents a critical step for understanding their physiological functions. The methods of direct RNA sequencing are quickly developing allowing for the precise location of internal RNA marks. This detection is, however, not quantitative and still presents detection limits. One of the biggest remaining challenges in the field is still the detection and quantification of m6A, m6Am, inosine, and m1A modifications of adenosine. The second intriguing and timely question remaining to be addressed is the extent to which individual marks are coregulated or potentially can affect each other. Here, we present a methodological approach to detect and quantify several key mRNA modifications in human total RNA and in mRNA, which is difficult to purify away from contaminating tRNA. We show that the adenosine demethylase FTO primarily targets m6Am marks in noncoding RNAs in HEK293T cells. Surprisingly, we observe little effect of FTO or ALKBH5 depletion on the m6A mRNA levels. Interestingly, the upregulation of ALKBH5 is accompanied by an increase in inosine level in overall mRNA.

• Keywords
• ADAR, ALKBH5, FTO, RNA editing, adenosine methylation, inosine,
• MeSH
• Adenosine * analogs & derivatives   metabolism   analysis   MeSH
• AlkB Homolog 5, RNA Demethylase genetics   metabolism   MeSH
• Chromatography, Liquid methods   MeSH
• Alpha-Ketoglutarate-Dependent Dioxygenase FTO genetics   metabolism   MeSH
• HEK293 Cells MeSH
• Inosine * metabolism   MeSH
• Liquid Chromatography-Mass Spectrometry MeSH
• Humans MeSH
• RNA, Messenger * genetics   chemistry   metabolism   MeSH
• Methylation MeSH
• RNA Processing, Post-Transcriptional MeSH
• Tandem Mass Spectrometry methods   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Adenosine * MeSH
• AlkB Homolog 5, RNA Demethylase MeSH
• ALKBH5 protein, human MeSH Browser
• FTO protein, human MeSH Browser
• Alpha-Ketoglutarate-Dependent Dioxygenase FTO MeSH
• Inosine * MeSH
• RNA, Messenger * MeSH
• N-methyladenosine MeSH Browser