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.
