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

A bstract. The occurrence of NAD⁺ as a non-canonical RNA cap has been demonstrated in diverse organisms. TIR domain-containing proteins present in all kingdoms of life act in defense responses and can have NADase activity that hydrolyzes NAD⁺. Here, we show that TIR domain-containing proteins from several bacterial and one archaeal species can remove the NAM moiety from NAD-capped RNAs (NAD-RNAs). We demonstrate that the deNAMing activity of AbTir (from Acinetobacter baumannii) on NAD-RNA specifically produces a cyclic ADPR-RNA, which can be further decapped in vitro by known decapping enzymes. Heterologous expression of the wild-type but not a catalytic mutant AbTir in E. coli suppressed cell propagation and reduced the levels of NAD-RNAs from a subset of genes before cellular NAD⁺ levels are impacted. Collectively, the in vitro and in vivo analyses demonstrate that TIR domain-containing proteins can function as a deNAMing enzyme of NAD-RNAs, raising the possibility of TIR domain proteins acting in gene expression regulation.

Various chemical modifications decorate RNA biomolecules and immensely expand the diversity of the transcriptome^1,2. RNA modifications can occur internally or terminally³, and may alter the molecular function, subcellular location, and stability of RNA⁴. Regarding the 5′-terminal RNA structure, besides the most common triphosphophate in prokaryotes and the N⁷-methylguanosine (m⁷G) cap in eukaryotes, the redox cofactor nicotinamide adenine dinucleotide (NAD⁺) has emerged as a non-canonical RNA cap structure in diverse organisms. These include prokaryotes such as Escherichia coli (E. coli)^5,6,7, Bacillus subtilis (B. subtilis)⁸, Streptomyces venezuelae (S. venezuelae)⁵, and Mycobacterium smegmatis⁹, and eukaryotes such as yeast¹⁰, mammalian cells¹¹, and plants^12,13,14,15. 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_nN), are also increasingly described^{3,17,18,19,20,21}.

The identity of NAD-capped RNAs (NAD-RNAs) varies in different species. 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^6,7,8,22. 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.

Thus far, two classes of NAD-RNA decapping enzymes have been identified in prokaryotic or eukaryotic organisms²⁴ (Supplementary Fig. 1). Class-I decapping enzymes, such as the E. coli Nudix hydrolase NudC, cleave the pyrophosphate bond within the NAD cap to liberate the nicotinamide mononucleotide (NMN)⁶ (Supplementary Fig. 1). Another bacterial Nudix protein, BsRppH, catalyzes the decapping of NAD-RNAs in Gram-positive B. subtilis by releasing NMN⁸. Homologs of NudC in eukaryotic organisms, like Npy1 in yeast²⁵ and Nudt12/16 in mammalian cells²⁶, have also been validated as the decapping enzymes. Class-II decapping enzymes, such as the DXO/Rai1 family enzymes in yeast, Arabidopsis, and mammals, remove the entire NAD cap, producing monophosphorylated RNAs (p-RNAs)^11,25,27,28 (Supplementary Fig. 1). The 5′−3′ exoribonucleases Xrn1 and Rat1 in the yeast Saccharomyces cerevisiae also serve as NAD cap decapping (deNADding) enzymes, similar to DXO/Rai1, but primarily act on mitochondrial NAD-RNAs²⁹. Homologs of DXO/Rai1 have not been found in prokaryotic organisms. More recently, the human glycohydrolase CD38 has been shown to convert NAD-RNAs into ADP-ribose-capped RNAs by liberating nicotinamide (NAM) in vitro²⁴ (Supplementary Fig. 1), although its in vivo decapping activity still needs validation. It is worth noting that NudC and DXO/Rai1 can act on FAD-capped and dpCoA-capped RNAs as well^30,31,32 and are thus not specific to NAD-RNAs.