What exactly is NAD-modified RNA, and why is it important?

NAD-modified RNA refers to RNA molecules that have been modified with nicotinamide adenine dinucleotide (NAD) at their 5' end. This modification was discovered in bacteria and archaea, and later in eukaryotes, challenging previous assumptions about RNA structure. NAD is a redox coenzyme involved in various cellular processes. The NAD-RNA modification can affect RNA stability, translation, and interactions with other molecules. This process may be related to post-translational protein modification. This is significant because it suggests a direct link between cellular metabolism and RNA function, opening new avenues for understanding gene regulation and cellular signaling.

Can you explain the NAD-captureSeq protocol and its main steps?

The NAD-captureSeq protocol is a chemoenzymatic method designed to isolate and identify NAD-modified RNA from a pool of total RNA. This process uses high-throughput sequencing (NGS). The protocol involves treating total RNA with adenosine diphosphate (ADP)-ribosyl cyclase (ADPRC) to facilitate transglycosylation. The resulting alkynyl alcohol is biotinylated, allowing for enrichment via streptavidin-biotin interaction. After adapter sequences are attached, cDNA libraries are generated for NGS analysis. The protocol provides a means to comprehensively map the presence and location of NAD modifications on RNA molecules, which is essential for understanding their functional roles.

What is an m7G-cap, and what functions does it perform?

The m7G-cap is a methylated guanosine residue linked to the mRNA via an inverted 5'-5'-triphosphate bridge. It is found on the 5' end of viral and eukaryotic mRNAs. It influences crucial processes like splicing, 3'-polyadenylation, RNA stability, and protein synthesis initiation. Removal of the cap, known as decapping, converts the RNA into a 5'-monophosphorylated form, inhibiting translation initiation and triggering targeted RNA degradation by RNases. The m7G-cap is essential for the proper processing and translation of mRNA molecules in eukaryotic cells. This contrasts with NAD-modified RNA found in bacteria.

What are small regulatory RNAs (sRNAs), and what's the significance of finding them with NAD modifications?

Small regulatory RNAs (sRNAs) are non-coding RNA molecules that play a crucial role in regulating gene expression in bacteria. They typically function by binding to mRNA molecules, either promoting or inhibiting translation, or by affecting mRNA stability. The discovery that sRNAs can be NAD-modified suggests a novel layer of regulation, potentially linking their activity to cellular metabolism and redox state. The identification of NAD-modified sRNAs expands our understanding of the mechanisms by which bacteria adapt to environmental changes and control gene expression.

What does redox biochemistry have to do with RNA, and why is it relevant?

Redox biochemistry involves the study of oxidation-reduction reactions in biological systems, where electrons are transferred between molecules. NAD, as a redox coenzyme, plays a central role in these reactions. The discovery of NAD-modified RNA suggests a direct link between redox biochemistry and RNA processing. This means that the redox state of the cell can influence RNA function through NAD modification. It also suggests the roles of post-translational protein modification, and various signalling pathways. This connection opens new avenues for understanding how cellular metabolism and environmental stress can impact gene regulation and cellular signaling.

Surreal digital illustration of NAD-modified RNA.

NAD-Modified RNA: How Redox Biochemistry is Revolutionizing RNA Processing

Soraya Malik in Science & Nature September 2025 • 4 min read.

"Unlocking the Secrets of RNA Modification and Its Impact on Cellular Processes"

RNA, a cornerstone of life, deftly manages genetic information, bridging the gap between DNA storage and protein synthesis. But its role goes far beyond mere information transfer. Regulatory RNAs, ubiquitous across all life forms, orchestrate pivotal biological processes. While RNA's catalytic prowess in modern biology may seem limited, its regulatory influence is undeniably vast.

The functional versatility of RNA stands in stark contrast to its deceptively simple chemical makeup. Composed of just four nucleotides – adenosine, guanosine, cytidine, and uridine – RNA achieves remarkable complexity through chemical modifications at various positions on these canonical nucleobases. These modifications, varying in location, frequency, and chemical structure, introduce a second layer of coding known as the epitranscriptome.

To date, scientists have identified approximately 160 distinct RNA modifications. While most occur internally, the 5' and 3' ends of RNA display limited diversity. Recent research has illuminated the significance of modifications at the 5' end, particularly in eukaryotes and prokaryotes, where newly synthesized RNAs initially bear a triphosphate group at the +1 nucleotide. Depending on cellular processing, these 5'-triphosphate termini can transform into diphosphate, monophosphate, or hydroxyl functionalities.

Unveiling NAD-Modified RNA: A Novel Frontier in Redox Biochemistry

Surreal digital illustration of NAD-modified RNA.

In the mid-1970s, biochemical analyses revealed that certain viral and eukaryotic mRNAs possess a unique cap: a methylated guanosine residue linked to the mRNA via an inverted 5'-5'-triphosphate bridge (m7G-cap). This m7G-cap influences crucial processes like splicing, 3'-polyadenylation, RNA stability, and protein synthesis initiation. Removing the cap, known as decapping, converts the RNA into a 5'-monophosphorylated form, inhibiting translation initiation and triggering targeted RNA degradation by RNases.

Contrary to previous assumptions, primary transcripts in bacteria and archaea were believed to possess a 5'-triphosphate originating from the +1 nucleotide. Bacterial RNA caps were previously unknown, however, in 2009, mass spectrometry revealed that cofactors, such as the redox coenzyme nicotinamide adenine dinucleotide (NAD), can covalently bind to the 5' end of bacterial RNAs. Yet, the identity of these NAD-modified RNAs remained elusive.

NAD-captureSeq Protocol: Developed a NAD-specific chemoenzymatic method (NAD-captureSeq) for specifically enriching NAD-modified RNA from total RNA and identifying the enriched RNAs via high-throughput sequencing (NGS).
Key Steps: Isolation and enrichment of NAD-modified RNA, cDNA preparation and NGS, and validation of NGS hits. For enriching 5'-NAD-modified RNA, total RNA is treated with adenosine diphosphate (ADP)-ribosyl cyclase (ADPRC) from Aplysia californica.
Transglycosylation: The enzyme surprisingly catalyzes transglycosylation of NAD-RNA with alkynyl alcohols. The "clickable" alkyne residue, selectively introduced instead of nicotinamide, is then biotinylated via copper-catalyzed azide-alkyne cycloaddition.

The biotinylated, formerly NAD-modified RNA is then enriched via a streptavidin-biotin interaction. Subsequently, adapter sequences are attached to both ends of the enriched RNAs, and reverse transcription generates a cDNA library for NGS analysis. Comparing the sequencing data to the genome identifies the enriched RNAs. In 2015, the NAD-captureSeq protocol was first applied to Escherichia coli total RNA, revealing that small regulatory RNAs (sRNAs) and various mRNA fragments are NAD-modified at their 5' ends.

The Future of RNA Research

The discovery and characterization of NAD-modified RNA in prokaryotes and eukaryotes has unlocked a new frontier in molecular biology. Given NAD's central role in redox biochemistry, post-translational protein modification, and various signaling pathways, we can speculate whether NAD-RNA, alongside NAD, finds use in these processes. Further research promises to reveal the full extent of NAD-modified RNA's influence on cellular function.