Diadenosine Tetraphosphate (Ap<sub>4</sub>A) Serves as a 5′ RNA Cap in Mammalian Cells
Jiří František Potužník, Ondřej Nešuta, Anton Škríba, Barbora Voleníková, Maria‐Bianca Mititelu, Flaminia Mancini, Valentina Serianni, Henri Fernandez, Kristína Spustová, Jana Trylčová, Pavel Vopálenský, Hana Cahová
Abstract
Abstract The recent expansion of the field of RNA chemical modifications has changed our understanding of post‐transcriptional gene regulation. Apart from internal nucleobase modifications, 7‐methylguanosine was long thought to be the only eukaryotic RNA cap. However, the discovery of non‐canonical RNA caps in eukaryotes revealed a new niche of previously undetected RNA chemical modifications. We are the first to report the existence of a new non‐canonical RNA cap, diadenosine tetraphosphate (Ap 4 A), in human and rat cell lines. Ap 4 A is the most abundant dinucleoside polyphosphate in eukaryotic cells and can be incorporated into RNA by RNA polymerases as a non‐canonical initiating nucleotide (NCIN). Using liquid chromatography‐mass spectrometry (LC–MS), we show that the amount of capped Ap 4 A‐RNA is independent of the cellular concentration of Ap 4 A. A decapping enzyme screen identifies two enzymes cleaving Ap 4 A‐RNA,NUDT2 and DXO, both of which also cleave other substrate RNAs in vitro. We further assess the translatability and immunogenicity of Ap 4 A‐RNA and show that although it is not translated, Ap 4 A‐RNA is recognized as self by the cell and does not elicit an immune response, making it a natural component of the transcriptome. Our findings open a previously unexplored area of eukaryotic RNA regulation.