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 (FurNAD-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. FurNAD-RNAs allow for real-time quantification of decapping activity, parallelization, high-throughput screening and identification of novel decapping enzymes in vitro. Using FurNAD-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.
Highlights
The selective degradation of individual RNA species is a crucial component in the regulation of gene expression, enabling the cell to respond quickly to changing environmental conditions
Using FurNAD-RNAs, we discovered that the eukaryotic glycohydrolase CD38 processes nicotinamide adenine dinucleotide (NAD)-capped RNA in vitro into ADP-ribose-modified-RNA and nicotinamide and might act as a decapping enzyme in vivo
These authors found that the addition of 5-membered heterocycles, in particular pyrrole, to the 8-position of the adenine of NAD resulted in fluorogenic derivatives, in which the 8-(pyrrol-2-yl)-adenosine was the fluorophore, whereas the nicotinamide moiety acted as an efficient fluorescence quencher, likely by contact quenching
Summary
The selective degradation of individual RNA species is a crucial component in the regulation of gene expression, enabling the cell to respond quickly to changing environmental conditions. RNA capping is considered to be a hallmark of eukaryotic gene expression, in which a canonical 5’-N7-methyl guanosine cap (m7G) protects mRNA from degradation and modulates maturation, localization, and translation [1]. In 2009, the ubiquitous redox coenzyme nicotinamide adenine dinucleotide (NAD) was discovered as a new RNA modification in bacteria [2], and a few years later we identified the modified RNAs in Escherichia coli [3]. To identify NAD-capped RNAs and to enable their functional characterization, we developed a chemo-enzymatic capture approach (NAD captureSeq) [5]. Using this protocol, NAD-RNA conjugates were selectively enriched from E. coli total RNA and analysed by next-generation sequencing (NGS). The discovery of NAD-capped RNA [3] in the bacterium E. coli provided an unexpected link between redox biology, metabolism, and RNA processing [4], and represented the first description of a prokaryotic cap [6,7]
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