Detection of N ³ ‐Methylcytidine in RNA at Single Base Resolution Using Protoscript II

ABSTRACT

N ³ ‐methylcytidine (m ³ C), an RNA modification involving methylation of cytidine at the Watson‐Crick‐Franklin edge, has been identified in human tRNA, mitochondrial tRNA (mt‐tRNA), and mRNA. In tRNA and mt‐tRNA, it has been shown to play important roles in tRNA structure and translation. Here we describe a new approach which utilizes the high readthrough and mutation rates of Protoscript II (PSII) at m ³ C sites in combination with AlkB demethylation to detect m ³ C at single‐base resolution. We first validate the method on synthetic probes. Then, using this method, we successfully identify known m ³ C sites on tRNA and mt‐tRNA, as well as one new candidate tRNA m ³ C site. We further show that none of the m ³ C sites identified in cytosolic tRNA are likely METTL8 substrates. In contrast, the two m ³ C sites detected in mt‐tRNA are sensitive to METTL8 knockdown (KD) and overexpression (OE), in line with their previous characterization as METTL8 substrates. These results demonstrate that our method can be used to investigate changes in the m ³ C level in many sites using an unbiased approach. Overall, this study introduces a convenient high‐resolution method for m ³ C mapping with high readthrough rates, paving the way for future mechanistic studies on the biological function of m ³ C and its effector proteins.