Figure 4 - available via license: Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International
Content may be subject to copyright.
RNAP from M. smegmatis incorporates NAD + caps into Ms1 in vitro. Abortive transcription was used to monitor the relative affinities of ATP and NAD + as initiating substrates for RNAP at the Ms1 promoter (shown on top). The +1A and +2C positions are shown in bold in the red box. Representative primary data show the abortive transcript (NAD + -C*) formed by NAD + and the radiolabelled CTP (C*). The presence/absence of ATP and NAD + (each 200 µM) is indicated. The dashed line indicates that the lanes from the same gel were electronically assembled for presentation. The graph shows the quantification of the NAD + -C* dinucleotide, normalized to the transcription signal in the absence of ATP, which was set as 1 (lane 3). The bars show average values ± SD, n=3.
Source publication
Chemical modifications of RNA affect essential properties of transcripts, such as their translation, localization and stability. 5-end RNA capping with the ubiquitous redox cofactor nicotinamide adenine dinucleotide (NAD+) has been discovered in organisms ranging from bacteria to mammals. However, the hypothesis that NAD+ capping might be universal...
Context in source publication
Context 1
... + caps have also been detected in Streptomyces, a different Actinobacterial genus, but the exact NAD + -capped transcripts are unknown, and it will be of interest to identify whether sRNA in this genus carry this modification (66) Our search for promoters from which transcription initiated with NAD + in M. smegmatis revealed conserved motifs corresponding to -35 and -10 promoter elements recognized by sigma factors σ A and σ F . This is consistent with the fact that the Ms1 promoter can be transcribed in vitro by the RNAP holoenzyme containing σ A (Figure 4), and that it was previously observed that Ms1 accumulated after σ A overexpression (23). F was previously reported to be active under oxidative stress, heat shock, low pH and in stationary phase (67-69) and is also highly expressed in exponential phase (70). ...
Citations
... Studies in Bacillus subtilis showed that mRNA is increasingly capped with NAD in the late exponential growth phase. Depletion of the B. subtilis deNADing enzyme BsRppH positively or negatively affected the expression of 13% genes clearly connecting NAD-RNA to gene regulation [90]. NAD-RNA and its decapping machinery were also recently detected in archaea and mycobacteria, where the NAD cap probably serves as a degradation marker [74,91]. ...
RNA capping is a prominent RNA modification that influences RNA stability, metabolism, and function. While it was long limited to the study of the most abundant eukaryotic canonical m⁷G cap, the field recently went through a large paradigm shift with the discovery of non-canonical RNA capping in bacteria and ultimately all domains of life. The repertoire of non-canonical caps has expanded to encompass metabolite caps, including NAD, FAD, CoA, UDP-Glucose, and ADP-ribose, alongside alarmone dinucleoside polyphosphate caps, and methylated phosphate cap-like structures. This review offers an introduction into the field, presenting a summary of the current knowledge about non-canonical RNA caps. We highlight the often still enigmatic biological roles of the caps together with their processing enzymes, focusing on the most recent discoveries. Furthermore, we present the methods used for the detection and analysis of these non-canonical RNA caps and thus provide an introduction into this dynamic new field.
... As the adenosine moiety of NAD is not involved in this reaction, we speculated that elongation of the adenosine to long RNA chains (by means of regular 5′-3′ phosphodiester bonds) might be tolerated by ARTs, potentially leading to the formation of covalent RNA-protein conjugates (Fig. 1b). RNAs that have a 5′-NAD cap have previously been found in bacteria (including E. coli 3,10,11 ), archaea 12,13 and eukaryotes 5,[14][15][16][17][18][19] , with NAD-RNA concentrations ranging from 1.9 to 7.4 fmol µg −1 RNA 16 . This modification was observed in different types of RNA, including mRNA and small regulatory RNA (sRNA) 20 . ...
The mechanisms by which viruses hijack the genetic machinery of the cells they infect are of current interest. When bacteriophage T4 infects Escherichia coli, it uses three different adenosine diphosphate (ADP)-ribosyltransferases (ARTs) to reprogram the transcriptional and translational apparatus of the host by ADP-ribosylation using nicotinamide adenine dinucleotide (NAD) as a substrate1,2. NAD has previously been identified as a 5′ modification of cellular RNAs3–5. Here we report that the T4 ART ModB accepts not only NAD but also NAD-capped RNA (NAD–RNA) as a substrate and attaches entire RNA chains to acceptor proteins in an ‘RNAylation’ reaction. ModB specifically RNAylates the ribosomal proteins rS1 and rL2 at defined Arg residues, and selected E. coli and T4 phage RNAs are linked to rS1 in vivo. T4 phages that express an inactive mutant of ModB have a decreased burst size and slowed lysis of E. coli. Our findings reveal a distinct biological role for NAD–RNA, namely the activation of the RNA for enzymatic transfer to proteins. The attachment of specific RNAs to ribosomal proteins might provide a strategy for the phage to modulate the host’s translation machinery. This work reveals a direct connection between RNA modification and post-translational protein modification. ARTs have important roles far beyond viral infections⁶, so RNAylation may have far-reaching implications.
... In eukaryotes, various Nudix hydrolases (termed "NudT") capable of removing different caps from RNA in vitro have been identified (Abdelraheim et al., 2003;Song et al., 2010Song et al., , 2013Grudzien-Nogalska et al., 2019). NAD-capped RNA has been identified in bacteria and eukaryotes and recently also in the archaeal domain in Sulfolobus acidocaldarius and Haloferax volcanii (Chen et al., 2009;Cahová et al., 2015;Jiao et al., 2017;Walters et al., 2017;Ruiz-Larrabeiti et al., 2021;Gomes-Filho et al., 2022). Furthermore, methylated and non-methylated dinucleoside polyphosphates (Np n Ns) were identified at the 5′ ends of E. coli RNA (Hudeček et al., 2020) and most recently, ADPR-capped RNA was identified in human cells (Weixler et al., 2022). ...
Nudix hydrolases comprise a large and ubiquitous protein superfamily that catalyzes the hydrolysis of a nucleoside diphosphate linked to another moiety X (Nudix). Sulfolobus acidocaldarius possesses four Nudix domain-containing proteins (SACI_RS00730/Saci_0153, SACI_RS02625/Saci_0550, SACI_RS00060/Saci_0013/Saci_NudT5, and SACI_RS00575/Saci_0121). Deletion strains were generated for the four individual Nudix genes and for both Nudix genes annotated to encode ADP-ribose pyrophosphatases (SACI_RS00730, SACI_RS00060) and did not reveal a distinct phenotype compared to the wild-type strain under standard growth conditions, nutrient stress or heat stress conditions. We employed RNA-seq to establish the transcriptome profiles of the Nudix deletion strains, revealing a large number of differentially regulated genes, most notably in the ΔSACI_RS00730/SACI_RS00060 double knock-out strain and the ΔSACI_RS00575 single deletion strain. The absence of Nudix hydrolases is suggested to impact transcription via differentially regulated transcriptional regulators. We observed downregulation of the lysine biosynthesis and the archaellum formation iModulons in stationary phase cells, as well as upregulation of two genes involved in the de novo NAD⁺ biosynthesis pathway. Furthermore, the deletion strains exhibited upregulation of two thermosome subunits (α, β) and the toxin-antitoxin system VapBC, which are implicated in the archaeal heat shock response. These results uncover a defined set of pathways that involve archaeal Nudix protein activities and assist in their functional characterization.
... NADcapSeq transpired to be a decisive advance in assessing the function of these caps in RNA metabolism. Subsequent NADcapSeq analyses of yeast 8 , plant 9 , human 10 , and archaeal RNA 11,12 have led to the identification of a significant number of transcripts with a NAD cap and have established the presence of the NAD cap as a 5′ non-canonical cap in all three domains of life. ...
Accurate identification of NAD-capped RNAs is essential for delineating their generation and biological function. Previous transcriptome-wide methods used to classify NAD-capped RNAs in eukaryotes contain inherent limitations that have hindered the accurate identification of NAD caps from eukaryotic RNAs. In this study, we introduce two orthogonal methods to identify NAD-capped RNAs more precisely. The first, NADcapPro, uses copper-free click chemistry and the second is an intramolecular ligation-based RNA circularization, circNC. Together, these methods resolve the limitations of previous methods and allowed us to discover unforeseen features of NAD-capped RNAs in budding yeast. Contrary to previous reports, we find that 1) cellular NAD-RNAs can be full-length and polyadenylated transcripts, 2) transcription start sites for NAD-capped and canonical m 7 G-capped RNAs can be different , and 3) NAD caps can be added subsequent to transcription initiation. Moreover, we uncovered a dichotomy of NAD-RNAs in translation where they are detected with mito-chondrial ribosomes but minimally on cytoplasmic ribosomes indicating their propensity to be translated in mitochondria.
... A typical example of a noncanonical RNA cap is nicotinamide adenine dinucleotide (NAD), which has been detected in all domains of life. [2][3][4][5] Other caps, such as NADH (a reduced form of NAD), 6 coenzyme A (CoA), 7 and flavin adenine dinucleotide (FAD), 8 have been detected only in some organisms. Recently, we have discovered an entirely new class of RNA caps in bacteria with the structure of dinucleoside polyphosphates (Np n Ns). 9 The presence of free Np n Ns molecules in cells of various organisms has been known more than 50 years. ...
... While some of the small molecules are known to be in vitro substrates of these NudiX enzymes, the entire set of recently discovered noncanonical caps has not yet been tested as free compounds. Therefore, we screened all the noncanonical RNA caps in their free form as substrates for the four NudiX proteins ( [5][6][7][8]. To better observe the selectivity of the enzyme towards small free molecules, we decreased its concentration to 50 nM ( Supplementary Fig. 9). ...
Recent discoveries of various noncanonical RNA caps, such as dinucleoside polyphosphates (Np n N), coenzyme A (CoA), and nicotinamide adenine dinucleotide (NAD) in all domains of life have led to a revision of views on RNA cap function and metabolism. Enzymes from the NudiX family capable of hydrolyzing a polyphosphate backbone attached to a nucleoside are the strongest candidates for degradation of noncanonically capped RNA. The model plant organism Arabidopsis thaliana encodes as many as 28 NudiX enzymes. For most of them, only in vitro substrates in the form of small molecules are known. In our study, we focused on four A. thaliana NudiX enzymes (AtNUDT6, AtNUDT7, AtNUDT19 and AtNUDT27), and we studied whether these enzymes can cleave RNA capped with Np n Ns (Ap2-5A, Gp3-4G, Ap3-5G, m7Gp3G, m7Gp3A), CoA, ADP-ribose, or NAD(H). While AtNUDT19 preferred NADH-RNA over other types of capped RNA, AtNUDT6 and AtNUDT7 preferentially cleaved Ap4A-RNA. The most powerful decapping enzyme was AtNUDT27, which cleaved almost all types of capped RNA at a tenfold lower concentration than the other enzymes. We also compared cleavage efficiency of each enzyme on free small molecules with RNA capped with corresponding molecules. We found that AtNUDT6 prefers free Ap4A, while AtNUDT7 preferentially cleaved Ap4A-RNA. These findings show that NudiX enzymes may act as RNA-decapping enzymes in A. thaliana and that other noncanonical RNA caps such as Ap4A and NADH should be searched for in plant RNA.
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.
Conspectus
Ribonucleic acid (RNA) is composed primarily of four canonical building blocks. In addition, more than 170 modifications contribute to its stability and function. Metabolites like nicotinamide adenine dinucleotide (NAD) were found to function as 5′-cap structures of RNA, just like 7-methylguanosine (m⁷G). The identification of NAD-capped RNA sequences was first made possible by NAD captureSeq, a multistep protocol for the specific targeting, purification, and sequencing of NAD-capped RNAs, developed in the authors’ laboratory in the year 2015. In recent years, a number of NAD-RNA identification protocols have been developed by researchers around the world. They have enabled the discovery and identification of NAD-RNAs in bacteria, archaea, yeast, plants, mice, and human cells, and they play a key role in studying the biological functions of NAD capping. We introduce the four parameters of yield, specificity, evaluability, and throughput and describe to the reader how an ideal NAD-RNA identification protocol would perform in each of these disciplines. These parameters are further used to describe and analyze existing protocols that follow two general methodologies: the capture approach and the decapping approach. Capture protocols introduce an exogenous moiety into the NAD-cap structure in order to either specifically purify or sequence NAD-capped RNAs. In decapping protocols, the NAD cap is digested to 5′-monophosphate RNA, which is then specifically targeted and sequenced. Both approaches, as well as the different protocols within them, have advantages and challenges that we evaluate based on the aforementioned parameters. In addition, we suggest improvements in order to meet the future needs of research on NAD-modified RNAs, which is beginning to emerge in the area of cell-type specific samples. A limiting factor of the capture approach is the need for large amounts of input RNA. Here we see a high potential for innovation within the key targeting step: The enzymatic modification reaction of the NAD-cap structure catalyzed by ADP-ribosyl cyclase (ADPRC) is a major contributor to the parameters of yield and specificity but has mostly seen minor changes since the pioneering protocol of NAD captureSeq and needs to be more stringently analyzed. The major challenge of the decapping approach remains the specificity of the decapping enzymes, many of which act on a variety of 5′-cap structures. Exploration of new decapping enzymes or engineering of already known enzymes could lead to improvements in NAD-specific protocols. The use of a curated set of decapping enzymes in a combinatorial approach could allow for the simultaneous detection of multiple 5′-caps. The throughput of both approaches could be greatly improved by early sample pooling. We propose that this could be achieved by introducing a barcode RNA sequence before or immediately after the NAD-RNA targeting steps. With increased processing capacity and a potential decrease in the cost per sample, protocols will gain the potential to analyze large numbers of samples from different growth conditions and treatments. This will support the search for biological roles of NAD-capped RNAs in all types of organisms.
