Detection and sequencing of Ap₂N-capped RNAs in human cells

It was long believed that the eukaryotic RNA was primarily capped with 7-methylguanosine (m⁷G). However, the expanding field of noncanonical RNA caps recently revealed the presence of other structures (nicotinamide adenine dinucleotide, flavin adenine dinucleotide, dinucleoside polyphosphates) at the 5' end of various mRNAs and noncoding RNAs. We report here on the detection and quantification of a new type of RNA cap-diadenosine diphosphate (Ap₂A) and adenosine guanosine diphosphate (Ap₂G)-on mammalian RNA. We have established a liquid chromatography-mass spectrometry (LC-MS) method to measure Ap₂N-RNA levels and observed its increase under oxidative stress. To identify Ap₂N-capped RNAs, we have developed a 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. We applied this method to human short RNA and identified a subset of tRNA-derived species as candidate Ap₂N-capped RNAs. Among these, Ap₂N-RNA Seq revealed previously uncharacterized 3' tRNA fragments consistent with Ap₂A capping at their 5' ends. This expands current understanding of tRNA capping and processing and broadens the toolkit for studying the RNA life cycle.