Sequencing of polyA+ RNA from shCtrl and shMETTL8 HeLa for m3C site identification

In tRNA, it has been shown to play important roles in tRNA structure and translation. m3C remains understudied in mRNA due to its markedly lower levels, the lack of high-resolution techniques to reproducibly map its location, and under-characterization of its effector proteins on this RNA type.

Additionally, METTL8 has been identified as a writer of m3C on mt-tRNA, but has also been shown to have isoforms with nuclear localization. Here, we apply a new approach which utilizes the high readthrough and mutation rates of Protoscript II (PSII) at m3C sites in combination with AlkB demethylation to map m3C at single-base resolution in polyA-purified RNA from HeLa cells either with METTL8-targeting shRNA or control shRNA to assess how the method works on mRNA and to investigate which, if any, of the sites change with METTL8 knockdown.