Identification of NAD-RNAs and ADPR-RNA decapping in the archaeal model organismsSulfolobus acidocaldariusandHaloferax volcanii

Abstract

NAD is a coenzyme central to metabolism that was also found to serve as a 5′-terminal cap of bacterial and eukaryotic RNA species. The presence and functionality of NAD-capped RNAs (NAD-RNAs) in the archaeal domain remain to be characterized in detail. Here, by combining LC-MS and NAD captureSeq methodology, we quantified the total levels of NAD-RNAs and determined the identity of NAD-RNAs in the two model Archaea,Sulfolobus acidocaldariusandHaloferax volcanii. A complementary differential RNA-Seq (dRNA-Seq) analysis revealed that NAD transcription start sites (NAD-TSS) correlate with well-defined promoter regions and often overlap with primary transcription start sites (pTSS). The population of NAD-RNAs in the two archaeal organisms shows clear differences, withS. acidocaldariuspossessing more capped small non-coding RNAs (sncRNAs) and leader sequences. The NAD-cap did not prevent 5′→3′ exonucleolytic activity by the RNase Saci-aCPSF2. To investigate enzymes that facilitate the removal of the NAD-cap, four Nudix proteins ofS. acidocaldariuswere screened. None of the recombinant proteins showed NAD decapping activity. Instead, the activity of the Nudix protein Saci_NudT5 was triggered by the incubation of NAD-RNAs at elevated temperatures. Hyperthermophilic environments promote the thermal degradation of NAD into the toxic product ADPR. Incorporating NAD into NAD-RNAs and regulating ADPR-RNA decapping by Saci_NudT5 is proposed to provide additional layers of maintaining stable NAD levels in archaeal cells.

A viral ADP-ribosyltransferase attaches RNA chains to host proteins

“…1b). RNAs harbouring a 5’-NAD-cap were recently found in bacteria, including E. coli 3,10,11 , archaea 12,13 , and higher organisms 5,14–19 , where NAD-RNA concentrations range from 1.9 to 7.4 fmol/μg RNA 16 . The modification was observed in different RNA types, including messenger RNAs (mRNAs) and small regulatory RNAs (sRNAs) 20 .…”

Molecular Biology of Archaea - 2022

“…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).…”

Transcriptome profiling of Nudix hydrolase gene deletions in the thermoacidophilic archaeon Sulfolobus acidocaldarius

“…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).…”

“…The Nudix family is known for displaying a large substrate range, especially in vitro, encompassing canonical and oxidized nucleotides, nucleotide sugars, dinucleotide coenzymes, diadenosine polyphosphates and capped RNAs, as well as non-nucleotide substrates such as inositol pyrophosphates (McLennan, 2013;Srouji et al, 2017). While the Nudix protein Saci_ NudT5 demonstrated ADPR-RNA decapping ability in vitro, the other proteins' activities remain elusive (Gomes-Filho et al, 2022). To this end, this study presents the impact of Nudix hydrolase absence on the transcriptome of S. acidocaldarius.…”