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One or more keywords matched the following properties of Base-resolution sequencing of m6A in RNA
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abstract ? DESCRIPTION (provided by applicant): Reversible chemical modifications on DNA and histones play critical roles in regulating gene expression in eukaryotes. Prior to our work, no example of reversible chemical modifications on RNA that could affect gene expression had been shown. In 2011, we discovered the first RNA demethylase, FTO, a protein belonging to the AlkB family iron- and 2-ketoglutarate (2-KG)-dependent dioxygenases and which is associated with human fat mass obesity. FTO catalyzes oxidative demethylation of the most prevalent internal modifications of mammalian messenger RNA (mRNA) and other nuclear RNA, N6-methyladenosine (m6A). This result, taken together with subsequent transcriptome-wide mapping of m6A, has revived research interest in the investigation of mRNA modifications. We have also characterized the methyltransferase core complex as well as proteins that can selectively recognize m6A-modified mRNA; the binding of m6A-containing mRNA by a family of the reader proteins affects the translation status and lifetime of the target mRNA. While research is ongoing to investigate the functional roles of m6A in various biological processes in laboratories around the world, we have yet to overcome a significant technology hurdle for the study of m6A in RNA: at present no high-throughput sequencing method exists that can detect the exact locations of m6A and reveal the modification percentage of m6A at each site. In the current application, we propose two new methods for the transcriptome-wide, base-resolution sequencing of m6A: i) ADAR-mediated adenosine deamination that differentiates unmodified A from m6A; ii) methyltransferase-assisted chemical labeling of adenosine to identify unmodified A from m6A. Both methods convert unmodified A in RNA into a different base during reverse transcription (RT) and subsequent amplification. The presence of the methyl group on m6A hinders the conversion, thereby allowing us to differentiate A from m6A in sequencing. These methods will be validated in selected biological systems and will be used to investigate potential roles of m6A that may differentiate maternal from zygotic mRNA during maternal to zygotic transition using zebrafish as a model. We believe the availability of these methods will provide enabling tools to future research of m6A in RNA.
label Base-resolution sequencing of m6A in RNA
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  • m6A