Identification of NAD ⁺ capped mRNAs in Saccharomyces cerevisiae

Abstract

RNAs besides tRNA and rRNA contain chemical modifications, including the recently described 5′ nicotinamide-adenine dinucleotide (NAD + ) RNA in bacteria. Whether 5′ NAD-RNA exists in eukaryotes remains unknown. We demonstrate that 5′ NAD-RNA is found on subsets of nuclear and mitochondrial encoded mRNAs in Saccharomyces cerevisiae. NAD-mRNA appears to be produced cotranscriptionally because NAD-RNA is also found on pre-mRNAs, and only on mitochondrial transcripts that are not 5′ end processed. These results define an additional 5′ RNA cap structure in eukaryotes and raise the possibility that this 5′ NAD + cap could modulate RNA stability and translation on specific subclasses of mRNAs.T he number and prevalence of known chemical modifications on mRNAs have dramatically increased in the past several years (1). Quantification of these modification events suggests they occur in many RNAs (2, 3). Importantly, several of these modifications have functional consequences (4-6). For example, the presence of a single N 6 -methyladenosine within the 5′ UTR of an mRNA increases translation initiation (4). In addition, the methylation status of cytosine residues within the 3′ UTR of the p16(INK4) human mRNA affects mRNA stability (6). Due to the increasing sensitivity of RNA sequencing (RNA-Seq) and small-molecule mass spectrometry, it is reasonable to hypothesize that many novel chemical modifications within mRNAs remain to be discovered.One modification recently identified in bacteria is 5′ nicotinamide-adenine dinucleotide (NAD + )-linked RNA (7, 8). Because canonical bacterial RNAs contain a 5′ triphosphate terminus, addition of NAD + to the 5′ end of RNAs represents a rudimentary "capping" mechanism, perhaps designed to impart specific properties for these RNAs by granting them a more structurally complex 5′ end. Consistent with this idea, NAD + addition to RNAs appears to occur during transcription initiation (9), as opposed to the more complex eukaryote 5′ capping, which occurs after transcription has commenced (10). Because the NAD + modification defines the 5′ end of NAD-RNAs, this modification can affect RNA stability in Escherichia coli (7, 9).For decades, 5′ end classification and study of eukaryotic mRNAs have been restricted to canonical 7-methylguanosine (m 7 G) "caps" and their methylated variants (11). The m 7 G cap modulates numerous facets of mRNA metabolism, including stability (12, 13), translation (14, 15), and export (16). The importance of this modification is underscored by the substantial cellular machinery dedicated to its addition and removal (17,18). Thus, mRNAs containing noncanonical 5′ termini may have distinct properties and be subject to alternative metabolic events.Described herein is the identification of NAD-RNAs in the eukaryote Saccharomyces cerevisiae. Examples of NAD-RNAs in S. cerevisiae include nuclear encoded mRNAs for ribosomal proteins, as well as some mitochondrial encoded transcripts. Our data suggest that the NAD + moiety is added during initiation in both nuclear and mitoc...

5′ End Nicotinamide Adenine Dinucleotide Cap in Human Cells Promotes RNA Decay through DXO-Mediated deNADding

“…Moreover, while this manuscript was under consideration, Saccharomyces cerevisiae was also reported to contain NAD + -capped RNA (Walters et al, 2017), indicating a wider prevalence of NAD + capped RNAs in eukaryotic cells. Importantly, the deNADding activity of SpRai1 and KlDxo1 proteins (Figure 7) suggests the DXO family of proteins are general deNADding enzymes in eukaryotes.…”

Mitochondrial-epigenetic crosstalk in environmental toxicology

“…NAD+ is also a substrate for class III histone deacetylases, also known as sirtuins; since low glucose allows accumulation of NAD+, starvation activates sirtuins to deacetylate histones and DNA binding proteins to condense chromatin and reduce gene expression [1]. In addition, NAD+ was recently identified as a 5′ mRNA modification, or rudimentary cap, in bacteria during transcription initiation, perhaps imparting additional structural complexity for regulation and affecting RNA stability [62]. Recently, this modification was identified in a eukaryote, S. cerevisiae , and data suggest that the moiety is added during initiation in both nuclear and mitochondrial transcription [62].…”

“…In addition, NAD+ was recently identified as a 5′ mRNA modification, or rudimentary cap, in bacteria during transcription initiation, perhaps imparting additional structural complexity for regulation and affecting RNA stability [62]. Recently, this modification was identified in a eukaryote, S. cerevisiae , and data suggest that the moiety is added during initiation in both nuclear and mitochondrial transcription [62]. …”

RNA Capping by Transcription Initiation with Non-canonical Initiating Nucleotides (NCINs): Determination of Relative Efficiencies of Transcription Initiation with NCINs and NTPs

“…However, recently it has been established that adenine-containing cofactors, including nicotinamide adenine dinucleotide (NAD + ), reduced nicotinamide adenine dinucleotide (NADH), and 3’-desphospho-coenzyme A (dpCoA), can serve as alternative initiating substrates (‘non-canonical initiating nucleotides’; NCINs), yielding NCIN-capped RNA products that have distinctive 5’-end structures, stabilities, and translation efficiencies (Figures 1B–1C; Bird et al , 2016; Barvik et al , 2016; Jiao et al , 2017; Walters et al , 2017). It further has been established that the relative efficiencies of NCIN-mediated initiation vs. NTP-mediated initiation are determined by promoter sequence (Bird et al , 2016).…”