RNA 5'-modification with NAD+/NADH (oxidized/reduced nicotinamide adenine dinucleotide) has been found in bacteria, eukaryotes and viruses. 5'-NAD is incorporated into RNA by RNA polymerases (RNAPs) during the initiation of synthesis. It is unknown (i) which factors and physiological conditions permit substantial NAD incorporation into RNA in vivo and (ii) how 5'-NAD impacts gene expression and the fate of RNA in bacteria. Here we show in Escherichia coli that RNA NADylation is stimulated by low cellular concentration of the competing substrate ATP, and by weakening ATP contacts with RNAP active site. Additionally, RNA NADylation may be influenced by DNA supercoiling. RNA NADylation does not interfere with posttranscriptional RNA processing by major ribonuclease RNase E. It does not impact the base-pairing between RNAI, the repressor of plasmid replication, and its antisense target, RNAII. Leaderless NADylated model mRNA cI-lacZ is recognized by the 70S ribosome and is translated with the same efficiency as triphosphorylated cI-lacZ mRNA. Translation exposes the 5'-NAD of this mRNA to de-capping by NudC enzyme. We suggest that NADylated mRNAs are rapidly degraded, consistent with their low abundance in published datasets. Furthermore, we observed that ppGpp inhibits NudC de-capping activity, contributing to the growth phase-dependency of NADylated RNA levels.

Centre for Bacterial Cell Biology Biosciences Institute Faculty of Medical Sciences Newcastle University Newcastle upon Tyne NE2 4AX UK

Institute of Microbiology of the Czech Academy of Sciences Vídeňská 1083 142 20 Prague Czech Republic

Zobrazit více v PubMed

Cahova H., Winz M.L., Hofer K., Nubel G., Jaschke A.. NAD captureSeq indicates NAD as a bacterial cap for a subset of regulatory RNAs. Nature. 2015; 519:374–377. PubMed

Wang J., Alvin Chew B.L., Lai Y., Dong H., Xu L., Balamkundu S., Cai W.M., Cui L., Liu C.F., Fu X.Y.et al. .. Quantifying the RNA cap epitranscriptome reveals novel caps in cellular and viral RNA. Nucleic Acids Res. 2019; 47:e130. PubMed PMC

Kowtoniuk W.E., Shen Y., Heemstra J.M., Agarwal I., Liu D.R.. A chemical screen for biological small molecule-RNA conjugates reveals CoA-linked RNA. Proc. Natl. Acad. Sci. U.S.A. 2009; 106:7768–7773. PubMed PMC

Gomes-Filho J.V., Breuer R., Morales-Filloy H.G., Pozhydaieva N., Borst A., Paczia N., Soppa J., Hofer K., Jaschke A., Randau L.. Identification of NAD-RNA species and ADPR-RNA decapping in archaea. Nat. Commun. 2023; 14:7597. PubMed PMC

Julius C., Yuzenkova Y.. Bacterial RNA polymerase caps RNA with various cofactors and cell wall precursors. Nucleic Acids Res. 2017; 45:8282–8290. PubMed PMC

Bird J.G., Zhang Y., Tian Y., Panova N., Barvik I., Greene L., Liu M., Buckley B., Krasny L., Lee J.K.et al. .. The mechanism of RNA 5' capping with NAD+, NADH and desphospho-CoA. Nature. 2016; 535:444–447. PubMed PMC

Zhang H., Zhong H., Wang X., Zhang S., Shao X., Hu H., Yu Z., Cai Z., Chen X., Xia Y.. Use of NAD tagSeq II to identify growth phase-dependent alterations in E. coli RNA NAD(+) capping. Proc. Natl. Acad. Sci. U.S.A. 2021; 118:e2026183118. PubMed PMC

Frindert J., Zhang Y., Nubel G., Kahloon M., Kolmar L., Hotz-Wagenblatt A., Burhenne J., Haefeli W.E., Jaschke A.. Identification, biosynthesis, and decapping of NAD-capped RNAs in B. subtilis. Cell Rep. 2018; 24:1890–1901. PubMed

Ares-Arroyo M., Rocha E.P.C., Gonzalez-Zorn B.. Evolution of ColE1-like plasmids across gamma-proteobacteria: from bacteriocin production to antimicrobial resistance. PLoS Genet. 2021; 17:e1009919. PubMed PMC

Fozo E.M., Kawano M., Fontaine F., Kaya Y., Mendieta K.S., Jones K.L., Ocampo A., Rudd K.E., Storz G.. Repression of small toxic protein synthesis by the sib and OhsC small RNAs. Mol. Microbiol. 2008; 70:1076–1093. PubMed PMC

Miyakoshi M., Okayama H., Lejars M., Kanda T., Tanaka Y., Itaya K., Okuno M., Itoh T., Iwai N., Wachi M.. Mining RNA-seq data reveals the massive regulon of GcvB small RNA and its physiological significance in maintaining amino acid homeostasis in Escherichia coli. Mol. Microbiol. 2022; 117:160–178. PubMed PMC

Bennett B.D., Kimball E.H., Gao M., Osterhout R., Van Dien S.J., Rabinowitz J.D.. Absolute metabolite concentrations and implied enzyme active site occupancy in Escherichia coli. Nat. Chem. Biol. 2009; 5:593–599. PubMed PMC

Grudzien-Nogalska E., Bird J.G., Nickels B.E., Kiledjian M.. NAD-capQ” detection and quantitation of NAD caps. RNA. 2018; 24:1418–1425. PubMed PMC

Sharma S., Yang J., Grudzien-Nogalska E., Shivas J., Kwan K.Y., Kiledjian M.. Xrn1 is a deNADding enzyme modulating mitochondrial NAD-capped RNA. Nat. Commun. 2022; 13:889. PubMed PMC

Jiao X., Doamekpor S.K., Bird J.G., Nickels B.E., Tong L., Hart R.P., Kiledjian M.. 5' End nicotinamide adenine dinucleotide cap in Human cells promotes RNA decay through DXO-mediated deNADding. Cell. 2017; 168:1015–1027. PubMed PMC

Hofer K., Li S., Abele F., Frindert J., Schlotthauer J., Grawenhoff J., Du J., Patel D.J., Jaschke A.. Structure and function of the bacterial decapping enzyme NudC. Nat. Chem. Biol. 2016; 12:730–734. PubMed PMC

Mackie G.A. Ribonuclease E is a 5'-end-dependent endonuclease. Nature. 1998; 395:720–723. PubMed

Kim S.K., Lormand J.D., Weiss C.A., Eger K.A., Turdiev H., Turdiev A., Winkler W.C., Sondermann H., Lee V.T.. A dedicated diribonucleotidase resolves a key bottleneck for the terminal step of RNA degradation. eLife. 2019; 8:e46313. PubMed PMC

Fortes P., Inada T., Preiss T., Hentze M.W., Mattaj I.W., Sachs A.B.. The yeast nuclear cap binding complex can interact with translation factor eIF4G and mediate translation initiation. Mol. Cell. 2000; 6:191–196. PubMed

Zheng X., Hu G.Q., She Z.S., Zhu H.. Leaderless genes in bacteria: clue to the evolution of translation initiation mechanisms in prokaryotes. Bmc Genomics [Electronic Resource]. 2011; 12:361. PubMed PMC

Orlova M., Newlands J., Das A., Goldfarb A., Borukhov S.. Intrinsic transcript cleavage activity of RNA polymerase. Proc. Natl. Acad. Sci. U.S.A. 1995; 92:4596–4600. PubMed PMC

Kulbachinskiy A., Mustaev A.. Region 3.2 of the sigma subunit contributes to the binding of the 3'-initiating nucleotide in the RNA polymerase active center and facilitates promoter clearance during initiation. J. Biol. Chem. 2006; 281:18273–18276. PubMed

Julius C., Riaz-Bradley A., Yuzenkova Y.. RNA capping by mitochondrial and multi-subunit RNA polymerases. Transcription. 2018; 9:292–297. PubMed PMC

Atlas R.M. Handbook of Microbiological Media. 2010; 4th edn.Washington, D.C.; Boca Raton, FL: ASM Press; CRC Press/Taylor & Francis.

Putt K.S., Hergenrother P.J.. An enzymatic assay for poly(ADP-ribose) polymerase-1 (PARP-1) via the chemical quantitation of NAD(+): application to the high-throughput screening of small molecules as potential inhibitors. Anal. Biochem. 2004; 326:78–86. PubMed

Tomizawa J. Control of ColE1 plasmid replication: the process of binding of RNA I to the primer transcript. Cell. 1984; 38:861–870. PubMed

Xu F.F., Gaggero C., Cohen S.N.. Polyadenylation can regulate ColE1 type plasmid copy number independently of any effect on RNAI decay by decreasing the interaction of antisense RNAI with its RNAII target. Plasmid. 2002; 48:49–58. PubMed

Castro-Roa D., Zenkin N.. In vitro experimental system for analysis of transcription-translation coupling. Nucleic Acids Res. 2012; 40:e45. PubMed PMC

Pedersen K., Zavialov A.V., Pavlov M.Y., Elf J., Gerdes K., Ehrenberg M.. The bacterial toxin RelE displays codon-specific cleavage of mRNAs in the ribosomal A site. Cell. 2003; 112:131–140. PubMed

Yokogawa T., Ohno S., Nishikawa K.. Incorporation of 3-azidotyrosine into proteins through engineering yeast tyrosyl-tRNA synthetase and its application to site-selective protein modification. Methods Mol. Biol. 2010; 607:227–242. PubMed

Dove S.L., Hochschild A.. A bacterial two-hybrid system based on transcription activation. Methods Mol. Biol. 2004; 261:231–246. PubMed

Wiggs J.L., Bush J.W., Chamberlin M.J.. Utilization of promoter and terminator sites on bacteriophage T7 DNA by RNA polymerases from a variety of bacterial orders. Cell. 1979; 16:97–109. PubMed

Camps M. Modulation of ColE1-like plasmid replication for recombinant gene expression. Recent Pat DNA Gene Seq. 2010; 4:58–73. PubMed PMC

Moriya T., Kawamata A., Takahashi Y., Iwabuchi Y., Kanoh N.. An improved fluorogenic NAD(P)+ detection method using 2-acetylbenzofuran: its origin and application. Chem. Commun. (Camb.). 2013; 49:11500–11502. PubMed

Schneider D.A., Gourse R.L.. Relationship between growth rate and ATP concentration in Escherichia coli: a bioassay for available cellular ATP. J. Biol. Chem. 2004; 279:8262–8268. PubMed

Zhou Y., Wang L., Yang F., Lin X., Zhang S., Zhao Z.K.. Determining the extremes of the cellular NAD(H) level by using an Escherichia coli NAD(+)-auxotrophic mutant. Appl. Environ. Microb. 2011; 77:6133–6140. PubMed PMC

Klein W.L., Boyer P.D.. Energization of active transport by Escherichia coli. J. Biol. Chem. 1972; 247:7257–7265. PubMed

Igloi G.L., Kossel H.. Use of boronate-containing gels for electrophoretic analysis of both ends of RNA molecules. Methods Enzymol. 1987; 155:433–448. PubMed

Nubel G., Sorgenfrei F.A., Jaschke A.. Boronate affinity electrophoresis for the purification and analysis of cofactor-modified RNAs. Methods. 2017; 117:14–20. PubMed

Jin D.J., Gross C.A.. Mapping and sequencing of mutations in the Escherichia coli rpoB gene that lead to rifampicin resistance. J. Mol. Biol. 1988; 202:45–58. PubMed

Severinov K., Soushko M., Goldfarb A., Nikiforov V.. Rifampicin region revisited. New rifampicin-resistant and streptolydigin-resistant mutants in the beta subunit of Escherichia coli RNA polymerase. J. Biol. Chem. 1993; 268:14820–14825. PubMed

Richardson J.P. Initiation of transcription by Escherichia-Coli rna-polymerase from supercoiled and non-supercoiled bacteriophage Pm2 DNA. J. Mol. Biol. 1975; 91:477–487. PubMed

Mirkin S.M., Bogdanova E.S., Gorlenko Z.M., Gragerov A.I., Larionov O.A.. DNA supercoiling and transcription in Escherichia-Coli - influence of rna-polymerase mutations. Mol. Gen. Genet. 1979; 177:169–175. PubMed

Sudzinová P., Kambová M., Ramaniuk O., Benda M., Sanderová H., Krásny L.. Effects of DNA topology on transcription from rRNA promoters in. Microorganisms. 2021; 9:87. PubMed PMC

Mojica F.J., Higgins C.F.. In vivo supercoiling of plasmid and chromosomal DNA in an Escherichia coli hns mutant. J. Bacteriol. 1997; 179:3528–3533. PubMed PMC

Arold S.T., Leonard P.G., Parkinson G.N., Ladbury J.E.. H-NS forms a superhelical protein scaffold for DNA condensation. Proc. Natl. Acad. Sci. U.S.A. 2010; 107:15728–15732. PubMed PMC

Singh S.S., Singh N., Bonocora R.P., Fitzgerald D.M., Wade J.T., Grainger D.C.. Widespread suppression of intragenic transcription initiation by H-NS. Genes Dev. 2014; 28:214–219. PubMed PMC

Malmgren C., Wagner E.G., Ehresmann C., Ehresmann B., Romby P.. Antisense RNA control of plasmid R1 replication. The dominant product of the antisense rna-mrna binding is not a full RNA duplex. J. Biol. Chem. 1997; 272:12508–12512. PubMed

Furuichi Y., Shatkin A.J.. 5'-termini of reovirus mRNA: ability of viral cores to form caps post-transcriptionally. Virology. 1977; 77:566–578. PubMed

Resch A., Tedin K., Graschopf A., Haggard-Ljungquist E., Blasi U.. Ternary complex formation on leaderless phage mRNA. FEMS Microbiol. Rev. 1995; 17:151–157. PubMed

Andreev D., Hauryliuk V., Terenin I., Dmitriev S., Ehrenberg M., Shatsky I.. The bacterial toxin RelE induces specific mRNA cleavage in the A site of the eukaryote ribosome. RNA. 2008; 14:233–239. PubMed PMC

Moll I., Blasi U.. Differential inhibition of 30S and 70S translation initiation complexes on leaderless mRNA by kasugamycin. Biochem. Biophys. Res. Commun. 2002; 297:1021–1026. PubMed

Yarchuk O., Jacques N., Guillerez J., Dreyfus M.. Interdependence of translation, transcription and mRNA degradation in the lacZ gene. J. Mol. Biol. 1992; 226:581–596. PubMed

Proshkin S., Rahmouni A.R., Mironov A., Nudler E.. Cooperation between translating ribosomes and RNA polymerase in transcription elongation. Science. 2010; 328:504–508. PubMed PMC

Meyer S., Temme C., Wahle E.. Messenger RNA turnover in eukaryotes: pathways and enzymes. Crit. Rev. Biochem. Mol. Biol. 2004; 39:197–216. PubMed

Kiledjian M. Eukaryotic RNA 5'-end NAD(+) capping and DeNADding. Trends Cell Biol. 2018; 28:454–464. PubMed PMC

Taraseviciene L., Bjork G.R., Uhlin B.E.. Evidence for an RNA binding region in the Escherichia coli processing endoribonuclease RNase E. J. Biol. Chem. 1995; 270:26391–26398. PubMed

Bandyra K.J., Wandzik J.M., Luisi B.. Substrate recognition and autoinhibition in the Central ribonuclease RNase E. Mol. Cell. 2018; 72:275–285. PubMed PMC

Tomcsanyi T., Apirion D.. Processing enzyme ribonuclease E specifically cleaves RNA I. An inhibitor of primer formation in plasmid DNA synthesis. J. Mol. Biol. 1985; 185:713–720. PubMed

Ghosh S., Deutscher M.P.. Oligoribonuclease is an essential component of the mRNA decay pathway. Proc. Natl. Acad. Sci. U.S.A. 1999; 96:4372–4377. PubMed PMC

Vvedenskaya I.O., Bird J.G., Zhang Y., Zhang Y., Jiao X., Barvik I., Krasny L., Kiledjian M., Taylor D.M., Ebright R.H.et al. .. CapZyme-Seq comprehensively defines promoter-sequence determinants for RNA 5' Capping with NAD<sup/>. Mol. Cell. 2018; 70:553–564. PubMed PMC

Reigstad C.S., Hultgren S.J., Gordon J.I.. Functional genomic studies of uropathogenic Escherichia coli and host urothelial cells when intracellular bacterial communities are assembled. J. Biol. Chem. 2007; 282:21259–21267. PubMed

Wang B., Dai P., Ding D., Del Rosario A., Grant R.A., Pentelute B.L., Laub M.T.. Affinity-based capture and identification of protein effectors of the growth regulator ppGpp. Nat. Chem. Biol. 2019; 15:141–150. PubMed PMC

Balke V.L., Gralla J.D.. Changes in the linking number of supercoiled DNA accompany growth transitions in Escherichia coli. J. Bacteriol. 1987; 169:4499–4506. PubMed PMC

Lal A., Dhar A., Trostel A., Kouzine F., Seshasayee A.S., Adhya S.. Genome scale patterns of supercoiling in a bacterial chromosome. Nat. Commun. 2016; 7:11055. PubMed PMC

Balke V.L., Gralla J.D.. Changes in the linking number of supercoiled DNA accompany growth transitions in Escherichia-Coli. J. Bacteriol. 1987; 169:4499–4506. PubMed PMC

Court D.L., Gan J., Liang Y.H., Shaw G.X., Tropea J.E., Costantino N., Waugh D.S., Ji X.. RNase III: genetics and function; structure and mechanism. Annu. Rev. Genet. 2013; 47:405–431. PubMed PMC

Varik V., Oliveira S.R.A., Hauryliuk V., Tenson T.. HPLC-based quantification of bacterial housekeeping nucleotides and alarmone messengers ppGpp and pppGpp. Sci. Rep. 2017; 7:11022. PubMed PMC

Wolfram-Schauerte M., Pozhydaieva N., Grawenhoff J., Welp L.M., Silbern I., Wulf A., Billau F.A., Glatter T., Urlaub H., Jaschke A.et al. .. A viral ADP-ribosyltransferase attaches RNA chains to host proteins. Nature. 2023; 620:1054–1062. PubMed PMC

Malygin A.G., Shemyakin M.F.. Adenosine, NAD and FAD can initiate template-dependent RNA synthesis catalyzed by Escherichia coli RNA polymerase. FEBS Lett. 1979; 102:51–54. PubMed

Yarbrough L.R., Schlageck J.G., Baughman M.. Synthesis and properties of fluorescent nucleotide substrates for DNA-dependent rna-polymerases. J. Biol. Chem. 1979; 254:2069–2073. PubMed

Lamberte L.E., Baniulyte G., Singh S.S., Stringer A.M., Bonocora R.P., Stracy M., Kapanidis A.N., Wade J.T., Grainger D.C.. Horizontally acquired AT-rich genes in Escherichia coli cause toxicity by sequestering RNA polymerase. Nat. Microbiol. 2017; 2:16249. PubMed PMC