Diadenosine Tetraphosphate (Ap4 A) Serves as a 5' RNA Cap in Mammalian Cells.

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 (Ap4 A), in human and rat cell lines. Ap4 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 Ap4 A-RNA is independent of the cellular concentration of Ap4 A. A decapping enzyme screen identifies two enzymes cleaving Ap4 A-RNA,NUDT2 and DXO, both of which also cleave other substrate RNAs in vitro. We further assess the translatability and immunogenicity of Ap4 A-RNA and show that although it is not translated, Ap4 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.

Arabidopsis thaliana NudiXes have RNA-decapping activity.

Recent discoveries of various noncanonical RNA caps, such as dinucleoside polyphosphates (Np n N), coenzyme A (CoA), and nicotinamide adenine dinucleotide (NAD) in all domains of life have led to a revision of views on RNA cap function and metabolism. Enzymes from the NudiX family capable of hydrolyzing a polyphosphate backbone attached to a nucleoside are the strongest candidates for degradation of noncanonically capped RNA. The model plant organism Arabidopsis thaliana encodes as many as 28 NudiX enzymes. For most of them, only in vitro substrates in the form of small molecules are known. In our study, we focused on four A. thaliana NudiX enzymes (AtNUDT6, AtNUDT7, AtNUDT19 and AtNUDT27), and we studied whether these enzymes can cleave RNA capped with Np n Ns (Ap2-5A, Gp3-4G, Ap3-5G, m7Gp3G, m7Gp3A), CoA, ADP-ribose, or NAD(H). While AtNUDT19 preferred NADH-RNA over other types of capped RNA, AtNUDT6 and AtNUDT7 preferentially cleaved Ap4A-RNA. The most powerful decapping enzyme was AtNUDT27, which cleaved almost all types of capped RNA at a tenfold lower concentration than the other enzymes. We also compared cleavage efficiency of each enzyme on free small molecules with RNA capped with corresponding molecules. We found that AtNUDT6 prefers free Ap4A, while AtNUDT7 preferentially cleaved Ap4A-RNA. These findings show that NudiX enzymes may act as RNA-decapping enzymes in A. thaliana and that other noncanonical RNA caps such as Ap4A and NADH should be searched for in plant RNA.