Ruth Breuer’s research while affiliated with Philipps University of Marburg and other places

NAD is a coenzyme central to metabolism that also serves as a 5′-terminal cap for bacterial and eukaryotic transcripts. Thermal degradation of NAD can generate nicotinamide and ADP-ribose (ADPR). Here, we use LC-MS/MS and NAD captureSeq to detect and identify NAD-RNAs in the thermophilic model archaeon Sulfolobus acidocaldarius and in the halophilic mesophile Haloferax volcanii. None of the four Nudix proteins of S. acidocaldarius catalyze NAD-RNA decapping in vitro, but one of the proteins (Saci_NudT5) promotes ADPR-RNA decapping. NAD-RNAs are converted into ADPR-RNAs, which we detect in S. acidocaldarius total RNA. Deletion of the gene encoding the 5′−3′ exonuclease Saci-aCPSF2 leads to a 4.5-fold increase in NAD-RNA levels. We propose that the incorporation of NAD into RNA acts as a degradation marker for Saci-aCPSF2. In contrast, ADPR-RNA is processed by Saci_NudT5 into 5′-p-RNAs, providing another layer of regulation for RNA turnover in archaeal cells.

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.

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 acidocaldarius and Haloferax 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, with S. acidocaldarius possessing 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 of S. acidocaldarius were screened. None of the recombinant proteins showed NAD decapping activity. Instead, the Nudix protein Saci_NudT5 showed activity after incubating NAD-RNAs at elevated temperatures. Hyperthermophilic environments promote the thermal degradation of NAD into the toxic product ADPR. Incorporating NAD into RNAs and the regulation of ADPR-RNA decapping by Saci_NudT5 is proposed to provide additional layers of maintaining stable NAD levels in archaeal cells. Importance This study reports the first characterization of 5’-terminally modified RNA molecules in Archaea and establishes that NAD-RNA modifications, previously only identified in the other two domains of life, are also prevalent in the archaeal model organisms Sulfolobus acidocaldarius and Haloferax volcanii . We screened for NUDIX hydrolases that could remove the NAD-RNA cap and showed that none of these enzymes removed NAD modifications, but we discovered an enzyme that hydrolyzes ADPR-RNA. We propose that these activities influence the stabilization of NAD and its thermal degradation to potentially toxic ADPR products at elevated growth temperatures.

Post-transcriptional modifications fulfill many important roles during ribosomal RNA maturation in all three domains of life. Ribose 2'- O -methylations constitute the most abundant chemical rRNA modification and are, for example, involved in RNA folding and stabilization. In archaea, these modification sites are determined by variable sets of C/D box sRNAs that guide the activity of the rRNA 2'- O -methyltransferase fibrillarin. Each C/D box sRNA contains two guide sequences that can act in coordination to bridge rRNA sequences. Here, we will review the landscape of archaeal C/D box sRNA genes and their target sites. One focus is placed on the apparent accelerated evolution of guide sequences and the varied pairing of the two individual guides, which results in different rRNA modification patterns and RNA chaperone activities.