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.

• Klíčová slova
• Mass spectrometry, RNA, RNA capping, RNA sequencing, RNA structures,
• MeSH
• lidé MeSH
• messenger RNA * metabolismus   genetika   chemie   MeSH
• RNA čepičky * metabolismus   chemie   genetika   MeSH
• sekvenční analýza RNA * metody   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• přehledy MeSH
• Názvy látek
• messenger RNA * MeSH
• RNA čepičky * 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
• adenosintrifosfát metabolismus   MeSH
• bakteriální RNA metabolismus   genetika   MeSH
• DNA řízené RNA-polymerasy metabolismus   genetika   MeSH
• endoribonukleasy metabolismus   genetika   MeSH
• Escherichia coli * genetika   metabolismus   MeSH
• messenger RNA metabolismus   genetika   MeSH
• NAD * metabolismus   MeSH
• posttranskripční úpravy RNA MeSH
• proteiny z Escherichia coli metabolismus   genetika   MeSH
• proteosyntéza MeSH
• RNA čepičky * metabolismus   MeSH
• stochastické procesy MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• adenosintrifosfát MeSH
• bakteriální RNA MeSH
• DNA řízené RNA-polymerasy MeSH
• endoribonukleasy MeSH
• messenger RNA MeSH
• NAD * MeSH
• proteiny z Escherichia coli MeSH
• ribonuclease E MeSH Prohlížeč
• RNA čepičky * 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
• bakteriální RNA genetika   MeSH
• dinukleosidfosfáty genetika   MeSH
• DNA řízené RNA-polymerasy genetika   MeSH
• Escherichia coli genetika   MeSH
• hydrolasy působící na anhydridy kyselin metabolismus   MeSH
• konformace nukleové kyseliny MeSH
• metylace MeSH
• proteiny z Escherichia coli metabolismus   MeSH
• RNA čepičky genetika   MeSH
• stabilita RNA MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• ApaH protein, E coli MeSH Prohlížeč
• bakteriální RNA MeSH
• dinukleosidfosfáty MeSH
• DNA řízené RNA-polymerasy MeSH
• hydrolasy působící na anhydridy kyselin MeSH
• proteiny z Escherichia coli MeSH
• RNA čepičky MeSH
• RppH protein, E coli MeSH Prohlížeč

Nucleoside-containing metabolites such as NAD+ can be incorporated as 5' caps on RNA by serving as non-canonical initiating nucleotides (NCINs) for transcription initiation by RNA polymerase (RNAP). Here, we report CapZyme-seq, a high-throughput-sequencing method that employs NCIN-decapping enzymes NudC and Rai1 to detect and quantify NCIN-capped RNA. By combining CapZyme-seq with multiplexed transcriptomics, we determine efficiencies of NAD+ capping by Escherichia coli RNAP for ∼16,000 promoter sequences. The results define preferred transcription start site (TSS) positions for NAD+ capping and define a consensus promoter sequence for NAD+ capping: HRRASWW (TSS underlined). By applying CapZyme-seq to E. coli total cellular RNA, we establish that sequence determinants for NCIN capping in vivo match the NAD+-capping consensus defined in vitro, and we identify and quantify NCIN-capped small RNAs (sRNAs). Our findings define the promoter-sequence determinants for NCIN capping with NAD+ and provide a general method for analysis of NCIN capping in vitro and in vivo.

• Klíčová slova
• NudC, RNA capping, RNA polymerase, RNA-seq, Rai1, nicotinamide adenine dinucleotide, non-canonical initiating nucleotide, transcription, transcription initiation, transcription start site,
• MeSH
• DNA řízené RNA-polymerasy metabolismus   MeSH
• endoribonukleasy metabolismus   MeSH
• Escherichia coli genetika   metabolismus   MeSH
• exprese genu genetika   MeSH
• genetická transkripce genetika   MeSH
• NAD metabolismus   MeSH
• nukleotidy genetika   MeSH
• počátek transkripce fyziologie   MeSH
• promotorové oblasti (genetika) genetika   MeSH
• RNA čepičky genetika   MeSH
• transkriptom genetika   MeSH
• vysoce účinné nukleotidové sekvenování metody   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, N.I.H., Extramural MeSH
• Názvy látek
• DNA řízené RNA-polymerasy MeSH
• endoribonukleasy MeSH
• mRNA decapping enzymes MeSH Prohlížeč
• NAD MeSH
• nukleotidy MeSH
• RNA čepičky MeSH