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

ABSTRACT: The ubiquitous redox coenzyme nicotinamide adenine dinucleotide (NAD) acts as a noncanonical cap structure on prokaryotic and eukaryotic ribonucleic acids. Here we find that in budding yeast, NAD-RNAs are abundant (>1400 species), short (<170 nt), and mostly correspond to mRNA 5′-ends. The modification percentage of transcripts is low (<5%). NAD incorporation occurs mainly during transcription initiation by RNA polymerase II, which uses distinct promoters with a YAAG core motif for this purpose. Most NAD-RNAs are 3′-truncated. At least three decapping enzymes, Rai1, Dxo1, and Npy1, guard against NAD-RNA at different cellular locations, targeting overlapping transcript populations. NAD-mRNAs are not translatable in vitro. Our work indicates that in budding yeast, most of the NAD incorporation into RNA seems to be disadvantageous to the cell, which has evolved a diverse surveillance machinery to prematurely terminate, decap and reject NAD-RNAs.

ORGANISM(S): Saccharomyces cerevisiae

PROVIDER: GSE146368 | GEO | 2020/04/28

REPOSITORIES: GEO

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NAD capping of mRNAs is abundant but strongly guarded against in yeast

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Small RNAs in budding yeast

Project description:Small RNAs in budding yeast

| PRJNA123555 | ENA

AtDXO1 possesses deNADding and exonuclease activities and plays pleiotropic roles in gene regulation

Project description:RNA capping and decapping tightly coordinate with transcription, translation, and RNA decay to regulate gene expression. The DXO/Rai1 family of proteins have been implicated in mRNA decapping and decay, and mammalian DXO was recently found to also function as a decapping enzyme for NAD+-capped RNAs (NAD-RNA). The Arabidopsis genome contains a single gene encoding a DXO/Rai1 protein, AtDXO1. Here we show that AtDXO1 possesses both NAD-RNA decapping activity and 5’-3’ exonuclease activity and that the atdxo1 mutation increases the stability of NAD-RNAs. The atdxo1 mutant displays severe growth retardation, pale color, and multiple developmental defects. Transcriptome profiling analysis showed that the atdxo1 mutation leads to elevated expression of defense-related genes and down-regulation of photosynthesis genes. The autoimmunity phenotype of the mutant could be suppressed by either eds1 or npr1 mutation. Surprisingly, the various phenotypes associated with the atdxo1 mutant could be complemented by the enzymatically inactive AtDXO1. We found that AtDXO1 interacts with RNA cap methyltransferase (CMT). The atdxo1 mutation apparently enhances post-transcriptional gene silencing by elevating levels of siRNAs. Our study indicates that AtDXO1 regulates gene expression in various biological and physiological processes through its pleiotropic molecular functions in mediating RNA processing and decay.

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Yeast expression data from conditions that inhibit sirtuins

2010-05-11 | GSE18488 | GEO

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Project description:Sir2 is an NAD+-dependent histone deacetylase, and is the founding member of a large, phylogentically conserved, family of such deacetylases called the Sirtuins. The budding yeast, Saccharomyces cerevisiae, harbors 4 paralogs of Sir2, known as Hst1, Hst2, Hst3, and Hst4. Reducing the intracellular NAD+ concentration is inhibitory for the Sirtuins, and raising the intracellular nicotinamide (NAM) concentration is inhibitory. Microarray gene expression analysis was used to identify novel classes of yeast genes whose expression is altered when either NAD+ concentration is reduced or NAM is elevated. A subset of genes involved in thiamine biosynthesis was identified as being upregulated when Sir2 or Hst1 was inactivated. Several mutants in the NAD+ biosynthesis and salvage pathways were tested, along with WT and WT grown in the presence of 5 mM nicotinamide, which inhibits the Sirtuins. Duplicate cultures in rich YPD media were grown into log phase and then harvested. The deletion mutants npt1M-bM-^HM-^F, bna1M-bM-^HM-^F, tna1M-bM-^HM-^F, and pnc1M-bM-^HM-^F were compared to a wild-type (WT) strain. Additionally, a WT strain grown in the presence of 5 mM NAM was compared to WT without NAM. Total RNA was isolated and used for microarray hybridizations onto Affymetrix Yeast 2.0 Gene arrrays.

2010-05-10 | E-GEOD-18488 | biostudies-arrayexpress

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