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.

• Keywords
• Mass spectrometry, RNA, RNA capping, RNA sequencing, RNA structures,
• MeSH
• Humans MeSH
• RNA, Messenger * metabolism   genetics   chemistry   MeSH
• RNA Caps * metabolism   chemistry   genetics   MeSH
• Sequence Analysis, RNA * methods   MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Review MeSH
• Names of Substances
• RNA, Messenger * MeSH
• RNA Caps * MeSH

RNA 5'-modification with NAD+/NADH (oxidized/reduced nicotinamide adenine dinucleotide) has been found in bacteria, eukaryotes and viruses. 5'-NAD is incorporated into RNA by RNA polymerases (RNAPs) during the initiation of synthesis. It is unknown (i) which factors and physiological conditions permit substantial NAD incorporation into RNA in vivo and (ii) how 5'-NAD impacts gene expression and the fate of RNA in bacteria. Here we show in Escherichia coli that RNA NADylation is stimulated by low cellular concentration of the competing substrate ATP, and by weakening ATP contacts with RNAP active site. Additionally, RNA NADylation may be influenced by DNA supercoiling. RNA NADylation does not interfere with posttranscriptional RNA processing by major ribonuclease RNase E. It does not impact the base-pairing between RNAI, the repressor of plasmid replication, and its antisense target, RNAII. Leaderless NADylated model mRNA cI-lacZ is recognized by the 70S ribosome and is translated with the same efficiency as triphosphorylated cI-lacZ mRNA. Translation exposes the 5'-NAD of this mRNA to de-capping by NudC enzyme. We suggest that NADylated mRNAs are rapidly degraded, consistent with their low abundance in published datasets. Furthermore, we observed that ppGpp inhibits NudC de-capping activity, contributing to the growth phase-dependency of NADylated RNA levels.

• MeSH
• Adenosine Triphosphate metabolism   MeSH
• RNA, Bacterial metabolism   genetics   MeSH
• DNA-Directed RNA Polymerases metabolism   genetics   MeSH
• Endoribonucleases metabolism   genetics   MeSH
• Escherichia coli * genetics   metabolism   MeSH
• RNA, Messenger metabolism   genetics   MeSH
• NAD * metabolism   MeSH
• RNA Processing, Post-Transcriptional MeSH
• Escherichia coli Proteins metabolism   genetics   MeSH
• Protein Biosynthesis MeSH
• RNA Caps * metabolism   MeSH
• Stochastic Processes MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Adenosine Triphosphate MeSH
• RNA, Bacterial MeSH
• DNA-Directed RNA Polymerases MeSH
• Endoribonucleases MeSH
• RNA, Messenger MeSH
• NAD * MeSH
• Escherichia coli Proteins MeSH
• ribonuclease E MeSH Browser
• RNA Caps * MeSH

RNA capping is a prominent RNA modification that influences RNA stability, metabolism, and function. While it was long limited to the study of the most abundant eukaryotic canonical m7G cap, the field recently went through a large paradigm shift with the discovery of non-canonical RNA capping in bacteria and ultimately all domains of life. The repertoire of non-canonical caps has expanded to encompass metabolite caps, including NAD, FAD, CoA, UDP-Glucose, and ADP-ribose, alongside alarmone dinucleoside polyphosphate caps, and methylated phosphate cap-like structures. This review offers an introduction into the field, presenting a summary of the current knowledge about non-canonical RNA caps. We highlight the often still enigmatic biological roles of the caps together with their processing enzymes, focusing on the most recent discoveries. Furthermore, we present the methods used for the detection and analysis of these non-canonical RNA caps and thus provide an introduction into this dynamic new field.

• Keywords
• NAD, RNA, RNA cap, RNA modifications, dinucleoside polyphosphate, epitranscriptomics,
• MeSH
• Bacteria genetics   metabolism   MeSH
• Humans MeSH
• RNA Caps * metabolism   chemistry   MeSH
• RNA chemistry   metabolism   genetics   MeSH
• RNA Stability MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Review MeSH
• Names of Substances
• RNA Caps * MeSH
• RNA MeSH

It has been more than 50 years since the discovery of dinucleoside polyphosphates (NpnNs) and yet their roles and mechanisms of action remain unclear. Here, we show that both methylated and non-methylated NpnNs serve as RNA caps in Escherichia coli. NpnNs are excellent substrates for T7 and E. coli RNA polymerases (RNAPs) and efficiently initiate transcription. We demonstrate, that the E. coli enzymes RNA 5'-pyrophosphohydrolase (RppH) and bis(5'-nucleosyl)-tetraphosphatase (ApaH) are able to remove the NpnN-caps from RNA. ApaH is able to cleave all NpnN-caps, while RppH is unable to cleave the methylated forms suggesting that the methylation adds an additional layer to RNA stability regulation. Our work introduces a different perspective on the chemical structure of RNA in prokaryotes and on the role of RNA caps. We bring evidence that small molecules, such as NpnNs are incorporated into RNA and may thus influence the cellular metabolism and RNA turnover.

• MeSH
• RNA, Bacterial genetics   MeSH
• Dinucleoside Phosphates genetics   MeSH
• DNA-Directed RNA Polymerases genetics   MeSH
• Escherichia coli genetics   MeSH
• Acid Anhydride Hydrolases metabolism   MeSH
• Nucleic Acid Conformation MeSH
• Methylation MeSH
• Escherichia coli Proteins metabolism   MeSH
• RNA Caps genetics   MeSH
• RNA Stability MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• ApaH protein, E coli MeSH Browser
• RNA, Bacterial MeSH
• Dinucleoside Phosphates MeSH
• DNA-Directed RNA Polymerases MeSH
• Acid Anhydride Hydrolases MeSH
• Escherichia coli Proteins MeSH
• RNA Caps MeSH
• RppH protein, E coli MeSH Browser