Absolute transcription-start nucleotide m6Am stoichiometry quantification by CROWN-Seq
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ABSTRACT:
PROVIDER: PRJNA977234 | ENA |
REPOSITORIES: ENA
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Project description:The 5’ end RNA molecules transcribed by RNA polymerase II are known to be modified in human cells. Specifically, A-initiated transcripts can be methylated at both the 2’-O position in the ribose (2’-O-methylation, or cap-1) and the N6-position in the base (N6-methylation). Almost all A-initiated transcripts have the cap-1 modification, however, the N6-methylation is optional. As a result, A-initiated transcripts in the cell can be Am (cap-1 only) or m6Am (cap-1 and N6-methylation) modified. To study m6Am, we developed CROWN-seq, which simultaneously obtains the transcription-start information and the stoichiometry of N6-methyl. With CROWN-seq, we found that the previous m6Am mapping studies highly underestimated the diversity of the transcription-start site (nucleotide) of the transcriptome. In this study, we used CROWN-seq to reveal m6Am sites and stoichiometry among different human cell lines. We demonstrated that m6Am stoichiometry in mRNA is in general high, however, m6Am stoichiometry in snRNA, snoRNA, and their pseudogenes is more variable.
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Project description:Fat mass and obesity-associated protein (FTO) was discovered as the first RNA demethylase and mediates the demethylation of N6,2′-O-dimethyladenosine (m6Am), a prevalent RNA modification adjacent to the 5’ cap. Despite its significance, the regulation of the m6Am demethylase and the potential impact of virus infection on FTO remain largely unexplored. In this study, we show that the alphaherpesvirus pseudorabies virus (PRV) triggers phosphorylation of FTO and that this phosphorylation is caused by the viral serine/threonine protein kinase UL13. To test whether the phosphorylated FTO can result in different demethylation on snRNA, we performed CROWN-seq to quantify m6Am stoichiometry in cells.
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