A Novel NAD-RNA Decapping Pathway Discovered by Synthetic Light-Up NAD-RNAs.

Area of Science:

• Molecular Biology
• Biochemistry
• RNA Biology

Background:

• Transcriptome complexity relies on RNA processing and decay pathways.
• 5'-RNA cap removal initiates RNA degradation in eukaryotes.
• Nicotinamide adenine dinucleotide (NAD) is a newly identified 5'-RNA cap structure in prokaryotes and eukaryotes, distinct from the canonical N7-methyl guanosine cap.

Purpose of the Study:

• To introduce 8-(furan-2-yl)-substituted NAD-capped-RNA (^FurNAD-RNA) as a novel research tool.
• To enable the identification and characterization of new NAD-RNA decapping enzymes.
• To facilitate real-time quantification and high-throughput screening of decapping activity.

Main Methods:

• Synthesis and application of ^FurNAD-RNA compounds.
• Enzymatic reactions releasing a fluorescence quencher (nicotinamide or nicotinamide mononucleotide) upon decapping.
• In vitro assays for real-time quantification and high-throughput screening of decapping enzymes.

Main Results:

• ^FurNAD-RNAs enable real-time, parallelized, and high-throughput identification of NAD-RNA decapping enzymes.
• The eukaryotic glycohydrolase CD38 was identified as a novel NAD-RNA decapping enzyme.
• CD38 processes NAD-capped RNA in vitro into ADP-ribose-modified-RNA and nicotinamide.

Conclusions:

• ^FurNAD-RNA is a versatile tool for discovering and characterizing NAD-RNA decapping enzymes.
• CD38's role as a decapping enzyme suggests multiple pathways for NAD-RNA processing in eukaryotes.
• NAD-RNA decapping is an important and regulated process in eukaryotic organisms.