Identification of NAD-RNA species and ADPR-RNA decapping in Archaea

Abstract

NAD is a coenzyme central to metabolism that also serves as a 5′-terminal cap for bacterial and eukaryotic transcripts. Thermal degradation of NAD can generate nicotinamide and ADP-ribose (ADPR). Here, we use LC-MS/MS and NAD captureSeq to detect and identify NAD-RNAs in the thermophilic model archaeon Sulfolobus acidocaldarius and in the halophilic mesophile Haloferax volcanii. None of the four Nudix proteins of S. acidocaldarius catalyze NAD-RNA decapping in vitro, but one of the proteins (Saci_NudT5) promotes ADPR-RNA decapping. NAD-RNAs are converted into ADPR-RNAs, which we detect in S. acidocaldarius total RNA. Deletion of the gene encoding the 5′−3′ exonuclease Saci-aCPSF2 leads to a 4.5-fold increase in NAD-RNA levels. We propose that the incorporation of NAD into RNA acts as a degradation marker for Saci-aCPSF2. In contrast, ADPR-RNA is processed by Saci_NudT5 into 5′-p-RNAs, providing another layer of regulation for RNA turnover in archaeal cells.

Toll/interleukin-1 receptor (TIR) domain-containing proteins have NAD-RNA decapping activity

“…Recent studies have demonstrated the presence of NAD-RNAs in archaeal model organisms, with the highest NAD cap concentration reported so far 9 , 16 , thereby extending the existence of NAD-RNAs to all three domains of life. Given the widespread occurrence of NAD-RNAs and the conservation of TIR domains across various phyla 59 , 60 , we hypothesize that the deNAMing capacity of TIR domain might be evolutionarily conserved.…”

“…4c ), suggesting that these enzymes might work together to specifically decap NAD-RNAs. A recent report showed that ADPR-RNA exists in archaea and can be generated from NAD-RNA through the spontaneous loss of NAM 16 . Our findings suggest that TIR-catalyzed NAM removal is another mechanism to generate RNAs capped by ADPR or its variants (Fig.…”

“…These include prokaryotes such as Escherichia coli ( E. coli ) 5 – 7 , Bacillus subtilis ( B. subtilis ) 8 , Streptomyces venezuelae ( S. venezuelae ) 5 , and Mycobacterium smegmatis 9 , and eukaryotes such as yeast 10 , mammalian cells 11 , and plants 12 – 15 . Recent studies have provided evidence that NAD-capping of RNA also occurs in Archaea 9 , 16 , establishing the NAD cap modification as ubiquitous in all three domains of life. Additionally, other non-canonical RNA-caps, such as flavin adenine dinucleotide (FAD), desphospho-coenzyme A (dpCoA), uridine diphosphate glucose (UDP-Glc), uridine diphosphate N-acetyl glucosamine (UDP-GlcNAc), and dinucleotide polyphosphate (Np n N), are also increasingly described 3 , 17 – 21 .…”

“…In bacteria, the protein-coding mRNAs are the major constituents of the NAD-capped transcriptome, and some small regulatory RNAs also tend to carry the NAD cap 6 – 8 , 22 . NAD-RNAs in archaea and the eukaryotic kingdom also encompass various types of RNA species 9 , 16 , 23 , underscoring the importance of studying the biogenesis and function of NAD-RNAs.…”

T4 phage RNA is NAD-capped and alters the NAD-cap epitranscriptome ofEscherichia coliduring infection through a phage-encoded decapping enzyme

“…Therefore strain-promoted (copper-free) azide-alkyne cycloaddition (SPAAC) (9) and Nanopore sequencing have been applied to identify NAD-RNAs (10). Overall, based on the NAD captureSeq technology, NAD-RNAs have been identified in various bacteria (11,12), including Escherichia coli (5,10), archaea (13,14) and eukaryotes (9,15–21), highlighting the universal existence of this cofactor-capped RNA species (8).…”