New findings in the field of RNA modifications have shed light on the intricate world of RNA diversity and functionality. Recently, researchers have uncovered a non-canonical RNA cap structure in various organisms, including bacteria such as Escherichia coli (E. coli), Bacillus subtilis, and Streptomyces venezuelae, as well as eukaryotes like yeast, mammalian cells, and plants. The identification of the non-canonical RNA cap, i.e., nicotinamide adenine dinucleotide (NAD), in all three domains of life highlights its potential significance in RNA biology.

Studies have revealed the presence of different classes of enzymes involved in the decapping of NAD-capped RNAs (NAD-RNAs) in prokaryotic and eukaryotic organisms. Class-I decapping enzymes, such as NudC in E. coli and its homologs in yeast and mammalian cells, cleave the pyrophosphate bond within the NAD cap to release nicotinamide mononucleotide (NMN) (Figure 1) and generate a monophosphorylated RNAs (p-RNA, the adenine nucleotide from NAD cap is included). Thus, this decapping activity can be also named as deNMNing activity. On the other hand, Class-II decapping enzymes, like DXO/Rai1 family enzymes in yeast, Arabidopsis, and mammals, remove the entire NAD cap to produce p-RNA (the adenine nucleotide from the NAD cap is excluded), which was referred to as the deNADding activity (Figure 1). In this study (https://doi.org/10.1038/s41467-024-46499-y), we have discovered that Toll/interleukin-1 receptor (TIR) domain-containing proteins also possess decapping activity on NAD-RNAs, whereby they remove the NAM moiety from the RNA cap. Together with ADPRC and CD38 enzymes, it was designated as a third class of NAD-RNA decapping enzymes (Class-III) with deNAMing activity (Figure 1). This activity is dependent on a conserved catalytic glutamate (E) residue and is enhanced under specific conditions that promote protein oligomerization. The RNA product generated after treatment with CD38/ADPRC/TIR domain-containing proteins was identified as ADPR-RNA or cyclic ADPR-RNA (including cADPR-RNA and its variant v-cADPR-RNA)(Figure 1), which can be further decapped by NudC or DXO/Rai1 enzymes. Importantly, TIR domain-containing proteins exhibit a specific decapping activity towards NAD-RNAs, distinguishing them from other known decapping enzymes. These decapping enzymes might play a role in regulating the stability and function of NAD-RNAs in different species.

Moreover, the study demonstrates the functional role of a bacterial TIR domain-containing protein, AbTir, in regulating the levels of free NAD⁺ and NAD-RNAs in E. coli cells. Through SPAAC-NAD-Seq profiling, researchers have identified specific genes involved in "molecule transport process" and "oxidoreductase activity" as targets of AbTir-mediated deNAMing in E. coli, confirming the in vivo activity of TIR domain-containing proteins on NAD-RNAs. Additionally, the study expands the understanding of TIR domain proteins by showing that an archaeal TIR domain-containing protein, TcpA, also exhibits deNAMing activity on NAD-RNAs, extending the role of TIR domains to Archaea.

Overall, these findings unveil NAD-RNA deNAMing as a novel molecular function of TIR domains, implicating these proteins in the regulation of gene expression. The study not only broadens our knowledge of RNA modifications but also opens new avenues for investigating the diverse functions of RNA caps in cellular processes across different domains of life.