Diadenosine Tetraphosphate (Ap4A) Serves as a 5′ RNA Cap in Mammalian Cells

• Published in: Angewandte Chemie International Edition Vol. 63; no. 6; pp. e202314951 - n/a
• Main Authors: František Potužník, Jiří, Nešuta, Ondřej, Škríba, Anton, Voleníková, Barbora, Mititelu, Maria‐Bianca, Mancini, Flaminia, Serianni, Valentina, Fernandez, Henri, Spustová, Kristína, Trylčová, Jana, Vopalensky, Pavel, Cahová, Hana
• Format: Journal Article
• Language: English
• Published: Germany Wiley Subscription Services, Inc 05.02.2024
• Edition: International ed. in English
• Subjects: Animals; Ap4A; Ap4A-RNA; Cell lines; Diadenosine Tetraphosphate; Dinucleoside Phosphates - metabolism; Eukaryotes; Gene regulation; Humans; Immune response; Immune system; Immunogenicity; Liquid chromatography; Mammalian cells; Mammals - metabolism; Mass spectrometry; Mass spectroscopy; Nucleotides; Nudix Hydrolases; Phosphoric Monoester Hydrolases; Rats; Ribonucleic acid; RNA; RNA Cap; RNA Caps; RNA Modification; Substrates; Transcriptomes; Diadenosine Tetraphosphate; Ap4A; RNA Cap; RNA Modification; Ap4A-RNA
• ISSN: 1433-7851; 1521-3773; 1521-3773
• DOI: 10.1002/anie.202314951

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 (Ap4A), in human and rat cell lines. Ap4A 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 Ap4A‐RNA is independent of the cellular concentration of Ap4A. A decapping enzyme screen identifies two enzymes cleaving Ap4A‐RNA,NUDT2 and DXO, both of which also cleave other substrate RNAs in vitro. We further assess the translatability and immunogenicity of Ap4A‐RNA and show that although it is not translated, Ap4A‐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. Diadenosine tetraphosphate‐RNA (Ap4A‐RNA) is found in human and rat cell lines. Its amount is not dependent on the intracellular concentration of Ap4A. Ap4A‐RNA is decapped by two enzymes, NUDT2 and DXO. Even though Ap4A‐RNA is present in the same fraction as mRNA, it is not translated. However, it is recognized as self RNA by the innate immune system, as it does not elicit an immune response.