Illumina sequencing of mammalian short RNA capped with Ap2A.. Uncovering Ap2N-Capped Human tRNAs and Their Fragments with Ap2N-RNA Sequencing

It was long believed that the eukaryotic RNA was only capped with 7-methylguanosine (m7G). However, expanding field of non-canonical RNA caps recently revealed the presence of other structures (e.g. nicotinamide adenine dinucleotide) at the 5’ end of various mRNAs and non-coding RNAs. We report here on the discovery and quantification of adenosine nucleoside diphosphate (Ap2N) caps on mammalian RNA, the concentrations of which change under oxidative stress. To identify Ap2N-capped RNAs, we have developed a new sequencing method (Ap2N-RNA Seq) by leveraging the ability of truncated T4 RNA Ligase 2 to selectively ligate Ap2N-capped RNAs to an RNA adaptor. By applying this method to human short RNA, we have found multiple non-coding RNAs (mainly tRNAs) to be capped with Ap2N. Surprisingly, in some cases, we even observed Ap2A at the 5′ end of 3′ tRNA fragments. This report presents evidence of a previously unknown type of tRNA capping and processing.