The 5’-NAD cap of RNAIII modulates toxin production in Staphylococcus aureus isolates

Abstract

10 Nicotinamide adenosine dinucleotide (NAD) has been found to be covalently attached to the 5'-11ends of specific RNAs in many different organisms, but the physiological consequences of this 12 modification are largely unknown. Here we report the occurrence of several NAD-RNAs in the 13 opportunistic human pathogen Staphylococcus aureus. Most prominently, RNAIII, a central 14 quorum-sensing regulator of this bacterium's physiology, was found to be 5'-NAD-capped to a 15 significant extent. NAD incorporation efficiency into RNAIII was found to depend in vivo on the -16 1 position of the P3 promoter. Reduction of RNAIII's NAD content led to a decreased expression 17of alpha-and delta-toxins, resulting in reduced cytotoxicity of the modified strains. These effects 18to not seem to be due to changes in RNAIII's secondary structure upon NAD attachment, as 19indicated by largely unaltered patterns in in vitro chemical probing experiments. Our study 20represents a large step towards establishing a biological function of the 5'-NAD cap, which for 21 RNAIII in S. aureus is to modulate the expression of virulence factors. 22 2 Importance 23Numerous organisms, including bacteria, are endowed with a 5'-NAD cap in specific RNAs. While 24 the presence of the 5'-NAD cap modulates the stability of the modified RNA species, a significant 25 biological function and phenotype have not been assigned so far. Here, we show the presence of 26 a 5'-NAD cap in RNAIII from S. aureus, a dual-function regulatory RNA involved in quorum-sensing 27 processes and regulation of virulence factor expression. We also demonstrate that altering the 28 natural NAD modification ratio of RNAIII leads to a decrease in exotoxin production, thereby 29 modulating bacterium's virulence. Our work unveils a new layer of regulation of RNAIII and the 30 agr system that might be linked to the redox state of the NAD molecule in the cell. 31

Use of NAD tagSeq II to identify growth phase-dependent alterations in E. coli RNA NAD + capping

“…RNAs in prokaryotes were once thought to lack a cap and have only a 5′-triphosphorylated end from the initiation nucleotide (5). However, it has been recently reported that some RNAs in bacteria contain a nicotinamide adenine dinucleotide (NAD + ) moiety at their 5′ end (6)(7)(8)(9)(10). Later, eukaryotic organisms, including yeast, mammalian cells, and Arabidopsis plants, were also found to produce NAD-capped RNAs (NAD-RNAs) (11)(12)(13)(14).…”

Chemical biology of nucleic acids

“…So far, only the method for NAD-RNA sequencing had been developed. 4) Thanks to that, we know that NAD-RNA is present in all types of cells including bacteria, 4,5) humans, 6) plants 7) and Archaea 8) . Nevertheless, the role of the NAD-RNA cap is still unclear.…”

Extensive 5’-Surveillance Guards Against Non-Canonical NAD-Caps of Nuclear mRNAs in Yeast

“…The cap is hydrolyzed by various decapping enzymes (Belasco, 2010), thereby triggering RNA degradation. Recently, another type of 5'-cap structure was discovered, both in prokaryotes Frindert et al, 2018;Morales-Filloy et al, 2020) and eukaryotes (Jiao et al, 2017;Walters et al, 2017;Wang et al, 2019;Zhang et al, 2019), that is derived from the ubiquitous redox coenzyme NAD. While in human and plant cells a diverse landscape of NAD-RNAs was found, only 37 RNA species and low abundance were reported in budding yeast (Walters et al, 2017), questioning the biological significance of NAD capping in this organism.…”