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Free keywords:
ADP-ribosyl Cyclase 1/genetics/*metabolism
Adenosine/chemistry
Biochemistry/methods
Endoribonucleases/genetics/metabolism
Fluorescence
Kinetics
Membrane Glycoproteins/genetics/*metabolism
NAD/genetics/*metabolism
Oligonucleotides/chemical synthesis
RNA Caps/chemistry/genetics/*metabolism
Spectrometry, Fluorescence
*Decapping
*NAD metabolism
*NAD-capped RNA
*RNA modification
*fluorescent RNA
*glycohydrolase
*high-throughput screening
Abstract:
The complexity of the transcriptome is governed by the intricate interplay of transcription, RNA processing, translocation, and decay. In eukaryotes, the removal of the 5'-RNA cap is essential for the initiation of RNA degradation. In addition to the canonical 5'-N7-methyl guanosine cap in eukaryotes, the ubiquitous redox cofactor nicotinamide adenine dinucleotide (NAD) was identified as a new 5'-RNA cap structure in prokaryotic and eukaryotic organisms. So far, two classes of NAD-RNA decapping enzymes have been identified, namely Nudix enzymes that liberate nicotinamide mononucleotide (NMN) and DXO-enzymes that remove the entire NAD cap. Herein, we introduce 8-(furan-2-yl)-substituted NAD-capped-RNA ((Fur)NAD-RNA) as a new research tool for the identification and characterization of novel NAD-RNA decapping enzymes. These compounds are found to be suitable for various enzymatic reactions that result in the release of a fluorescence quencher, either nicotinamide (NAM) or nicotinamide mononucleotide (NMN), from the RNA which causes a fluorescence turn-on. (Fur)NAD-RNAs allow for real-time quantification of decapping activity, parallelization, high-throughput screening and identification of novel decapping enzymes in vitro. Using (Fur)NAD-RNAs, we discovered that the eukaryotic glycohydrolase CD38 processes NAD-capped RNA in vitro into ADP-ribose-modified-RNA and nicotinamide and therefore might act as a decapping enzyme in vivo. The existence of multiple pathways suggests that the decapping of NAD-RNA is an important and regulated process in eukaryotes.
