NADcapPro Seq and CircNAD –Methods for Accurate Profiling of NAD and Non-Canonical RNA Caps in Eukaryotes
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PROVIDER: PRJNA898308 | ENA |
REPOSITORIES: ENA
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Project description:Accurate identification of NAD-capped RNAs is essential for understanding their biological function. Previous transcriptome-wide methods used to profile NAD-capped RNAs contain inherent limitations of having hindered the accurate identification of NAD caps from eukaryotic RNAs. Herein we introduced two novel orthogonal methods to precisely identify NAD-capped RNAs. One is D-SPAAC, a copper-free click-chemistry-based approach, and the second is an intramolecular ligation-based circNAD to resolve implicit limitations of the previous methods, which enabled us to unravel unforeseen features of NAD RNAs in budding yeast. Contrary to previous reports, we find that 1) cellular NAD RNAs can be full-length and polyadenylated transcripts, 2) transcription start sites for NAD-capped and canonical m7G-capped RNAs are different, and 3) NAD caps can be added post-transcriptionally. Moreover, we uncovered a dichotomy of NAD RNAs in translation where NAD RNAs are detected with mitochondrial ribosomes but not cytoplasmic ribosomes indicating their propensity to be translated in mitochondria.
2023-03-17 | GSE217259 | GEO
Project description:We developed a Copper-free, strain-promoted azide-alkyne cycloaddition reaction (SPAAC) to capture NAD-RNAs without RNA degradation. We examined the specificity of CuAAC and SPAAC reactions towards NAD+ vs. m7G, and found that both prefer NAD+ but also act on m7G. We show that m7G-capped RNA can be immuno-depleted, allowing for the specific identification of NAD-RNA via the SPAAC reaction and sequencing, which we name SPAAC-NAD-seq. Subjecting Arabidopsis RNA to both the original NAD captureSeq and SPAAC-NAD-seq, we found that more NAD+-capped RNA was identified by the latter, particularly those with low abundance. This led to the discovery of new gene ontology terms such as starch biosynthsis, intracellular protein transport and response to cadmium stress associated with genes that produce NAD-RNA. Furthermore, reads were uniformly distributed along gene bodies, which suggested that SPAAC-NAD-seq retained full-length sequence information. SPAAC-NAD-seq enables specific and efficient discovery of NAD-RNA in prokaryotes, and when combined with m7G-RNA depletion, in eukaryotes.
2021-04-14 | GSE162114 | GEO
Project description: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.
2020-04-28 | GSE146368 | GEO
Project description:Extensive 5'-Surveillance Guards Against Non-Canonical NAD-Caps of Nuclear mRNAs in Yeast
| PRJNA610259 | ENA
Project description:SPAAC-NAD-seq, a sensitive and accurate method to profile NAD+-capped transcripts
| PRJNA680602 | ENA
Project description:Eukaryotic mRNAs generally possess an N7 methyl guanosine cap at their 5? end to promote their translation and stability. Here we demonstrate mammalian mRNAs can carry a 5'-end nicotinamide adenine dinucleotide (NAD+) cap. We further demonstrate fungal and mammalian noncanonical DXO family of decapping enzymes can efficiently remove NAD+ caps from mRNAs in vitro and cocrystal structures of DXO with 3´ phosphate NAD+ illuminates the molecular mechanism for the “deNADing” reaction. An NAD+ cap promotes mRNA decay in wild type mammalian cells and confers mRNA stability in the absence of DXO. Importantly, mammalian cells possess a capping mechanism that NAD+ caps a subset of intronic small nucleolar RNAs that are selectively enriched in DXO deficient cells. Our data establish NAD+ as a bona fide mammalian RNA cap and identifies the DXO proteins as potent deNADing enzymes that modulate the levels of NAD+-capped RNAs in cells.
2017-03-09 | GSE90884 | GEO
Project description:NAD besides its key role in cellular metabolism can serve as an alternative 5’ cap at several short non-coding RNAs. However, the function of the NAD cap remains elusive. Here, we investigate NAD capping of RNAs upon HIV-1 infection, which is associated with intracellular pellagra – depletion of NAD/NADH cellular pool. We applied NAD captureSeq on HIV-1 infected/noninfected cells and we revealed that four snRNAs (U1, U4ATAC, U5E and U7) and four snoRNAs (snord3G, snord102, snorA50A and snord3B) lost NAD cap upon HIV-1 infection. Interestingly, U1 snRNA was previously shown to be essential for HIV-1 replication. We provide evidence that the NAD cap reduces the stability of the U1 - HIV-1 pre-mRNA duplex. The importance of NAD RNA cap in HIV-1 infection was further supported by NAD decapping enzyme DXO overexpression, which led to increase in HIV-1 infectivity. This is the first example of NAD cap function in mammalian cells and suggests a general role of non-canonical RNA caps in antiviral innate immunity response.
2024-05-16 | GSE191019 | GEO
Project description:The ubiquitous redox coenzyme nicotinamide adenine dinucleotide (NAD) acts as a non-canonical 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.
| S-EPMC7606564 | biostudies-literature
Project description:The 5’ end of a eukaryotic mRNA generally has a methyl guanosine cap (m7G cap) that not only protects the mRNA from degradation but also mediates almost all other aspects of gene expression. Some RNAs in E. coli, yeast, and mammals were recently found to contain an NAD+ cap at their 5’ ends. Here we report development of a new method – NAD tagSeq – for transcriptome-wide identification and quantification of NAD+-capped RNAs (NAD-RNAs). The method uses first an enzymatic reaction and then a click chemistry reaction to label NAD-RNAs with a synthetic RNA tag. The tagged RNA molecules can be enriched and directly sequenced using the Oxford Nanopore sequencing technology. NAD tagSeq not only allows more accurate identification and quantification of NAD-RNAs but can also reveal sequences of whole NAD-RNA transcripts. Using NAD tagSeq, we found that NAD-RNAs in Arabidopsis are mostly produced from a few thousand protein-coding genes, with over 60% of them from fewer than 200 genes. The top 2,000 genes that were found to produce the highest numbers of NAD-RNAs were enriched in the gene ontology terms of responses to oxidative stress and other stresses, photosynthesis, and protein synthesis. For some Arabidopsis genes, over 10% of their transcripts could be NAD-capped. The NAD-RNAs in Arabidopsis have similar overall sequence structures to their canonical m7G-capped mRNAs. The identification and quantification of NAD-RNAs and revealing their sequence features provide essential steps toward understanding functions of NAD-RNAs.
2019-07-01 | GSE127755 | GEO
Project description:Recent findings regarding NAD+-capped RNAs (NAD-RNAs) indicate that prokaryotes and eukaryotes employ non-canonical RNA capping to regulate genes, a previously unrecognized mechanism. Two methods for transcriptome-wide analysis of NAD-RNAs, NAD captureSeq and NAD tagSeq, are based on copper-catalyzed azide-alkyne cycloaddition click chemistry reaction to label NAD-RNAs. However, copper can fragment RNA, interfering with the analyses. Here we report development of NAD tagSeq II, which uses copper-free, strain-promoted azide-alkyne cycloaddition for labeling NAD-RNAs, followed by identification of tagged RNA by direct RNA sequencing. Using this method, we compared NAD-RNA and total transcript profiles of E. coli cells in the exponential and stationary phases and identified hundreds of NAD-RNA species. For some genes, the majority of their transcripts were found as NAD-RNAs. Our study indicates that NAD-RNAs are preferentially produced from inducible genes in response to different growth conditions.
2021-04-21 | GSE153253 | GEO