Site-specific quantification of Adenosine-to-Inosine RNA editing by Endonuclease-Mediated qPCR

RNA molecules contain diverse modified nucleobases that play pivotal roles in numerous biological processes. Adenosine-to-inosine (A-to-I) RNA editing, one of the most prevalent RNA modifications in mammalian cells, is linked to a multitude of human diseases. To unveil the functions of A-to-I RNA editing, accurate quantification of inosine at specific sites is essential. In this study, we developed an endonuclease-mediated cleavage and real-time fluorescence quantitative PCR method for A-to-I RNA editing (EM-qPCR) to quantitatively analyze A-to-I RNA editing at a single site. By employing this method, we successfully quantified the levels of A-to-I RNA editing on various transfer RNA (tRNA) molecules at position 34 (I₃₄) in mammalian cells with precision. Subsequently, this method was applied to tissues from sleep-deprived mice, revealing a notable alteration in the levels of I₃₄ between sleep-deprived and control mice. The proposed method sets a precedent for the quantitative analysis of A-to-I RNA editing at specific sites, facilitating a deeper understanding of the biological implications of A-to-I RNA editing.