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.

Keywords: NAD-RNA; RNA modification; mitochondria; transcription.

Fig. 1.

ADPRC treatment enriches for a biochemical fraction that contains NAD-RNA. (A) Structure of NAD-capped RNA. (B) PAGE gel of pCp [5′-³²P]-labeled RNA of total yeast RNA that was incubated after selection either with or without treatment with ADPRC. (C) Similar to B, total yeast RNA was either treated with ADPRC or mock-treated, run on a Northern blot, and probed for polyA⁺ RNA using a poly(dT) DNA probe.

Fig. S1.

Generation of 5′ NAD-RNA. (A) Sequence of in vitro-transcribed RNA. Note the presence of a single A (+1) in the transcript. (B) PAGE analysis of in vitro transcribed RNA. The RNA indicated in A was transcribed with α–³²P-GTP. The reactions also contained either cold ATP (which would initiate with adenosine; A-RNA) or cold NAD⁺ (initiation with NAD⁺; NAD-RNA). These RNAs were then subjected to ADPRC/streptavidin selection (Materials and Methods).

Fig. S2.

NAD-Capture-Seq technical replicates are highly similar. Three-dimensional plots of technical triplicates from libraries prepared from indicated media conditions (YEPD or synthetic defined).

Fig. 2.

NAD-Capture-Seq identifies NAD-RNA in yeast. Abundance of ADPRC-treated RNAs vs. mock-treated samples in rich media (A) and synthetic defined media (B). Red triangles indicate mRNAs enriched at least twofold and statistically enriched with P values <0.05 using standard parameters in DESeq software. Black dots indicate nonenriched RNAs. (C, Top) Overlap of NAD-RNAs in a WT yeast strain grown in YEPD and synthetic media (P = 2.15e-11). (C, Bottom) List of NAD-RNAs found in both growth conditions in WT yeast. (D) NAD-RNAs (ADPRC⁺/ADPRC⁻) (red triangles, statistically enriched and greater than twofold change) plotted vs. total mRNA abundance.

Fig. 3.

NAD-mRNAs are encoded in both the nuclear and mitochondrial genomes. (A) Gene Ontology (GO) term analysis of yeast NAD-RNAs. (B) Twenty most highly enriched NAD-RNAs in WT yeast grown in synthetic media. (C) Gene tracks depicting RNA-Seq reads of two enriched nuclear encoded mRNAs (EGD2 and GPP1), a mitochondrial encoded mRNA (ATP9), and a nonenriched mRNA (PDC1). (D) Northern blots of mRNAs identified by NAD-capture as well as 7S rRNA control.

Fig. 4.

NAD-mRNAs contain NAD⁺ on their cognate pre-mRNAs. (A, Left) Introns of NAD-capped RNAs are also enriched in NAD-Capture-Seq. (A, Right) Introns of all mRNAs are not significantly enriched. (B, Left) Representative gene track of COF1 mRNA, enriched in NAD-Seq. The intron of COF1 is also enriched. (B, Right) Gene track of ECM33 mRNA, not enriched in NAD-Seq. The intron of ECM33 is also not enriched.

Fig. 5.

Non-5′ end-processed mitochondrially encoded RNAs selectively contain NAD⁺. (A) Table of mitochondrial RNAs showing their enrichment in RNA-Seq and 5′ end-processing properties. (B) Representative gene track and read density along the 21S rRNA. Read density shows a 5′ bias. (C) Gene track of RNA-Seq reads across the COX1 transcript. (D and E) Gene tracks showing the 5′ ends of the COX2 and ATP9 mitochondrial NAD-capped transcripts. Ellipses indicate that the gene continues.