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ABSTRACT:
PROVIDER: PRJNA1125588 | 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:As the most common mRNA cap, the m7G cap impacts the fate of an mRNA in eukaryotes. The metabolite and redox agent, nicotinamide adenine diphosphate (NAD+), can be used as an initiating nucleotide in RNA synthesis to result in NAD+-capped RNAs. Such RNAs have been identified in bacteria, yeast, and human cells, but it is not known whether they exist in plant transcriptomes. The functions of the NAD+ cap in RNA metabolism or translation are still poorly understood. Here, through NAD captureSeq, we show that NAD+- capped RNAs are widespread in Arabidopsis thaliana. NAD+-capped RNAs are predominantly messenger RNAs encoded by the nuclear and mitochondrial genomes but not the chloroplast genome. NAD-capped transcripts from the nuclear genome appear to be spliced and polyadenylated. Furthermore, although NAD+-capped transcripts constitute a small proportion of the total transcript pool from any gene, they are enriched in the polysomal fraction and associate with translating ribosomes. Our findings implicate the existence of as yet unknown mechanisms of translation initiation on NAD+-capped mRNAs. More importantly, our findings suggest that cellular metabolic and/or redox states may influence, and maybe regulated by, mRNA NAD capping.
2019-06-03 | GSE127002 | GEO
Project description:The hub metabolite, nicotinamide adenine dinucleotide (NAD), can be used as an initiating nucleotide in RNA synthesis to result in NAD-capped RNAs (NAD-RNA). Since NAD has been heightened as one of the most essential modulators in aging and various age-related diseases, its attachment to RNA might indicate a yet-to-be discovered mechanism that impacts adult life-course. However, the unknown identity of NAD-linked RNAs in adult and aging tissues has hindered functional studies. Here, we introduce ONE-seq method to identify the RNA transcripts that contain NAD cap. ONE-seq has been optimized to use only one-step chemo-enzymatic biotinylation, followed by streptavidin capture and the nudix phosphohydrolase NudC-catalyzed elution, to specifically recover NAD-capped RNAs for epitranscriptome and gene-specific analyses. Our data describes more than a thousand of previously unknown NAD-RNAs in the mouse liver and reveals epitranscriptome-wide dynamics of NAD-RNAs with age.ONE-seq empowers the identification of NAD-capped RNAs that are responsive to distinct physiological states, facilitating functional investigation into this modification.
2022-01-27 | GSE194271 | GEO
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:It has recently been shown that RNA Polymerase II transcription is far more extensive than previously thought, much of it not associated with protein-coding genes. To investigate this phenomenon, we determined the genome-wide landscape of RNA Polymerase II transcription initiation and elongation in C. elegans. We identify 73,500 distinct clusters of transcription initiation and find that initiation is often bidirectional. Strikingly, the majority of initiation events occur in regions with enhancer-like chromatin signatures. We also assign transcription initiation sites to 7691 protein coding genes, the majority previously unknown because of trans-splicing. Through mapping RNA PolII initiation (short capped RNAs) and elongation (long capped RNAs), we provide identification of transcription start sites.
2013-03-29 | E-GEOD-42819 | biostudies-arrayexpress
Project description:The 5´-end 7-methylguanosine cap structure has long been known as a signature feature of eukaryotic cellular and viral mRNAs that confers mRNA stability and efficient translation. Recent findings in diverse organisms have demonstrated that RNAs can additionally possess a non-canonical cap structure consisting of a nicotinamide adenosine dinucleotide (NAD+) at their 5´ end in place of m7G. It has been shown that 5´ end-NAD+ cap promotes rapid decay of the RNA at least in part by the DXO family of proteins in mammalian cells. This observation led to the hypothesis that mammalian cells harbor additional deNADding enzymes that may function in distinct pathways. Here we report Nudt12 efficiently removes NAD+ caps and functions as alternate cellular deNADding enzyme that targets NAD+-capped RNAs distinct from DXO. Importantly, with the use of an NAD-Cap Detection (NAD-CapD) approach that utilizes enzymatic properties to release intact NAD+/NADH from the 5´ end of NAD-capped cellular RNAs and a colorimetric NAD Quantitation to detect released NAD+/NADH, we can follow total cellular NAD+ cap levels. Removal of Nudt12 or DXO deNADding enzymes from cells significantly increased levels of NAD+-capped cellular RNAs. Moreover, fungal Rai1 and Dxo1, previously demonstrated to possess deNADding activity in vitro, can also function as deNADding enzymes in yeast cells. Double disruption of Rai1 and Dxo1 in yeast cells lead to accumulation of NAD+-capped RNAs, indicating that both enzymes function to clear NAD+ from the 5´ end of RNAs. Finally, our findings established that alterations in cellular NAD+ levels impact NAD+-capped RNA levels implying NAD+ capping is a modulated process that may be linked to the metabolic state of the cell.
2019-05-20 | GSE110801 | GEO
Project description:CoPRO adapts PRO-cap for paired end sequencing, and includes a total of three different libraries that were enriched for either capped nascent RNAs only, uncapped nascent RNAs only, or both. With paired end sequencing, each read tells us where an RNA polymerase molecule initiated, and then where its active site is located. Comparison of the libraries for different capping states allows us identify the precise location of pausing, and of where nascent RNAs become capped across the tens of thousands of initiation sites that we detect. The paired nature of the data enabled identification of sites of transcription initiation with unprecedented precision: we could use the pattern of pausing and elongation as a sensitive way of calling real initiation sites, and filter out termination by comparing capped and uncapped treatments. Because CoPRO maps nascent RNA, it is not affected by the post-transcriptional stability of the initiation and pausing events detected. Thus, it puts non-coding transcription (such as eRNAs and upstream divergent RNAs) on an equal footing with longer lived RNAs like mRNA and lncRNAs. With our comprehensive maps of transcription initiation, we are able to compare the architecture of initiation sites with other features of the genome. For this purpose, we chose human K562 cells for direct comparison with dozens of publically available genome-wide datasets.
2018-07-02 | GSE116472 | GEO
Project description:The hub metabolite, nicotinamide adenine dinucleotide (NAD), can be used as an initiating nucleotide in RNA synthesis to result in NAD-capped RNAs (NAD-RNA). We investigated the dynamics of NAD-modified epitranscriptome during human normal aging.
2023-12-20 | GSE226636 | GEO
Project description:The lack of a comprehensive map of transcription start sites in Toxoplasma gondii has impeded advances in decrypting the molecular mechanisms underlying the regulation of gene expression. Genome-wide approaches for mapping transcription initiation have been instrumental in defining the determinants of transcription initiation in a range of model eukaryotes. We used a protocol for 5′-end RNA sequencing, called RAMPAGE, to sequence the 5′-ends of capped mRNA from ME49 and RH tachyzoites, as well as from ME49 bradyzoites. This allowed us to generate maps of transcription initiation events at single-nucleotide resolution across the asexual stages of the parasite.
2021-01-22 | GSE159515 | GEO
Project description:RNA polymerase II transcripts receive a protective 5ʹ m7G cap early during transcription. An alternative cap can be acquired when RNA pol II initiation uses the redox cofactor nicotinamide adenine dinucleotide (NAD) to generate NAD-capped RNAs. The biology of mammalian NAD-RNAs, including its turnover and physiological roles are not completely understood. Here we identify NUDT12 as a cytosolic decapping enzyme for NAD-RNAs. Structural, biochemical and functional studies reveal how homodimerization modulates specificity of NUDT12 for NAD-RNAs as substrate. NUDT12 is localized within a few discrete cytoplasmic granules that are distinct from P-bodies. We identity Bleomycin hydrolase (BLMH) as a factor that associates with NUDT12 in a large ~600 kDa dodecamer complex, and we demonstrate that BLMH is required for correct localization of NUDT12 into these granules. This complex is functional in human cell cultures, as artificial tethering of BLMH to a reporter mRNA results in downregulation of reporter expression, similar to that seen with tethering of NUDT12. Analysis of mouse liver transcriptome from the Nudt12 knockout mouse reveals that a select set of RNAs are regulated, including a significant upregulation of circadian clock transcripts. Given that NAD biosynthesis is under circadian control, our study points to a physiological role for the cytosolic surveillance of NAD-RNAs.
2019-12-25 | GSE138019 | GEO