Coenzyme- and metabolite-linked RNAs as a new paradigm in epitranscriptomics

Projektinformationen

RNACoenzyme

ID Finanzhilfevereinbarung: 882789

DOI

10.3030/882789

EK-Unterschriftsdatum 19 Juni 2020

Startdatum 1 Oktober 2020

Enddatum 30 Juni 2026

Finanziert unter

• EXCELLENT SCIENCE - European Research Council (ERC)

Gesamtkosten

€ 2 500 000,00

EU-Beitrag

€ 2 500 000,00

Koordiniert durch

RUPRECHT-KARLS-UNIVERSITAET HEIDELBERG
Germany

Periodic Reporting for period 3 - RNACoenzyme (Coenzyme- and metabolite-linked RNAs as a new paradigm in epitranscriptomics)

Berichtszeitraum: 2023-10-01 bis 2025-03-31

Zusammenfassung vom Kontext und den Gesamtzielen des Projekts

RNA plays various important roles in biology, such as carrying messages, acting as catalysts, and regulating biological processes. In 2015, we discovered that a molecule called nicotinamide adenine dinucleotide (NAD), which is involved in cellular metabolism, can be attached to bacterial RNAs in a similar way to a cap found in more complex organisms. Since then, researchers have found similar NAD-RNAs in other organisms like eukaryotes and archaea. Enzymes were discovered that synthesize or break down NAD-RNAs.
NAD is just one example of many coenzymes and metabolic intermediates that carry a nucleotide moiety that is not directly involved in the catalyzed reaction. However, its conservation throughout evolution suggests it still has important functions. This led us to hypothesize that the presence of nucleotide moieties in coenzymes and metabolites allows cells to incorporate these compounds into specific RNAs. This linking of reactive organic components to RNA could help localize the RNAs to enzymes, receptors, membranes, or compartments, allowing them to sense environmental conditions or regulate the turnover and function of the RNAs and their targets. Therefore, the aim of this project is to investigate the scope and biological significance of coenzyme-linked RNAs in biology. plan to expand their current method for capturing NAD-RNAs to include other modified forms of NAD-RNAs, such as those with reduced or phosphorylated components. We will also develop new methods called CoenzymeSeq to identify cellular RNAs modified with other coenzymes and metabolites, such as coenzyme A, flavin, thiamine, and N-acetylglucosamine. We will apply these protocols to RNAs isolated from different organisms to explore the occurrence, abundance, and structural variety of such RNAs. Additionally, the project aims to uncover the biological importance and biosynthesis of selected modified RNAs, challenging existing knowledge in textbooks This research will establish new connections between gene regulation and metabolism in modern biology and reveal a previously unknown layer of information called epitranscriptomics. Furthermore, the project will influence our fundamental understanding of the evolution of metabolism and enzymatic catalysis.

Arbeit, die ab Beginn des Projekts bis zum Ende des durch den Bericht erfassten Berichtszeitraums geleistet wurde, und die wichtigsten bis dahin erzielten Ergebnisse

In the initial phase of our project, we made important advancements in our research. We improved our NAD captureSeq technique to investigate different variations of NAD-RNAs, including those with reduced, phosphorylated, deamidated, and depyridinated forms. To accomplish this, we created synthetic model RNAs and tested various enzymes involved in NAD metabolism and redox biochemistry. We applied these modified techniques to bacterial RNA samples to explore the presence and abundance of these different forms. Notably, we achieved significant progress in detecting NAD-RNAs that lacked nicotinamide components, including the exciting discovery of a covalent linkage to host target proteins induced by a viral enzyme during infection.

We also tested innovative captureSeq protocols that may enable the identification and sequencing of RNAs linked to other coenzymes and metabolites such as CoA, FAD, AThDP, and UDP-GlcNAc. These protocols were applied to total RNA extracted from E. coli and other sources, leading to exciting findings, including the discovery of a previously unknown sugar modification in bacterial RNA.

Moreover, we created several analytical tools utilizing mass spectrometry and affinity electrophoresis to accurately identify and measure the levels of RNA-linked coenzymes and metabolites.

In parallel, we initiated the testing and characterization of potential enzymes involved in the integration, conversion, and removal of RNA-linked coenzymes and metabolites. This progress places us in a favorable position to tackle the main objectives outlined in our research proposal during the upcoming two years.

Fortschritte, die über den aktuellen Stand der Technik hinausgehen und voraussichtliche potenzielle Auswirkungen (einschließlich der bis dato erzielten sozioökonomischen Auswirkungen und weiter gefassten gesellschaftlichen Auswirkungen des Projekts)

RNA biology is an exciting and rapidly evolving field in the life sciences. We now understand that RNA plays essential roles in catalysis, regulation, and signaling within living organisms. Recently, we made a groundbreaking discovery that challenged existing beliefs. We found that certain regulatory RNAs in bacteria are capped with a molecule called NAD+, which is a coenzyme involved in redox reactions. This finding connected RNA regulation with cellular metabolism and opened up new possibilities in RNA biology. Our discovery inspired extensive research across various laboratories, leading to significant publications in top scientific journals.

In line with our proposal in 2016, there is mounting evidence suggesting that other coenzymes and metabolites containing nucleotides are also incorporated into RNA. However, we have yet to identify these modified RNAs. In our current research proposal, we aim to go beyond studying NAD+ and investigate the comprehensive range and functions of RNA molecules linked to coenzymes and metabolites in all forms of life. This exploration has the potential to revolutionize our understanding of coenzyme functions, unveil a novel regulatory mechanism in present-day biology, shed light on the significance of nucleotide structures within these compounds, and explain why these molecules have been conserved over billions of years of evolution. Deciphering these roles and unraveling the mechanisms behind the formation of these new RNA modifications could reshape our understanding of RNA biology and potentially require updates in fundamental biochemistry textbooks. It may also lead to a reevaluation of the "molecular fossil" hypothesis concerning the origin of life.

Given the central roles of coenzymes and nucleotide-linked metabolites in cellular biochemistry, it becomes evident that exploring this topic from a medical perspective is crucial. These connections have direct implications for diseases and pathological conditions. For instance, NAD+ is a key player in redox metabolism across all biological systems. The discovery of NAD+-linked regulatory RNAs holds promise in understanding processes related to oxidative stress, aging, and vascular diseases. If we can establish causality in pathological processes, modified RNA species could offer new opportunities for long-term drug discovery endeavors.