The Mysterious World of Non-canonical Caps - What We Know and Why We Need New Sequencing Techniques.

It was long believed that viral and eukaryotic mRNA molecules are capped at their 5' end solely by the N7-methylguanosine cap, which regulates various aspects of the RNA life cycle, from its biogenesis to its decay. However, the recent discovery of a variety of non-canonical RNA caps derived from metabolites and cofactors - such as NAD, FAD, CoA, UDP-glucose, UDP-N-acetylglucosamine, and dinucleoside polyphosphates - has expanded the known repertoire of RNA modifications. These non-canonical caps are found across all domains of life and can impact multiple aspects of RNA metabolism, including stability, translation initiation, and cellular stress responses. The study of these modifications has been facilitated by sophisticated methodologies such as liquid chromatography-mass spectrometry, which have unveiled their presence in both prokaryotic and eukaryotic organisms. The identification of these novel RNA caps highlights the need for advanced sequencing techniques to characterize the specific RNA types bearing these modifications and understand their roles in cellular processes. Unravelling the biological role of non-canonical RNA caps will provide insights into their contributions to gene expression, cellular adaptation, and evolutionary diversity. This review emphasizes the importance of these technological advancements in uncovering the complete spectrum of RNA modifications and their implications for living systems.

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.