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Interplay between bacterial RNA 5'-termini deNADding protein NudC and DEAD-box RNA helicase CsdA in stress-responses

ABSTRACT: Nicotinamide adenine dinucleotide (NAD) "cap" recently discovered in bacteria could have similar functions as eukaryotic 7-methylguanine (m7G) "cap", which protects RNA 5'-terminus from degradation and mediates RNA interactions with proteins involved in its transportation, editing and translation. The only known 5'-NAD-RNA-interacting E. coli protein is Nudix superfamily hydrolase NudC which is responsible for this "cap" hydrolysis. We conducted the search for NudC-interacting proteins and discovered that in vivo and in vitro NudC interacts with RNA helicase DeaD. We also discovered using bacterial two hybrid system that N-terminal parts of both proteins are involved in this interaction, particularly the N-terminal domain and zinc finger motif of NudC and RecA1, RecA2 and dimerization domains of DeaD. Detailed investigation on the hydrolase interactome revealed NudC contacts with at least three proteins involved in formation of RNA degradosome complex, DeaD, Hfq and PNPase, under stress conditions raising the idea that NudC could also function within the RNA degradosome. On the other hand, NudC together with DeaD interacts with 70S ribosome proteins. Thus, NudC is closely related to protein synthesis in ribosomes. Finally, since at least six NudC-interacting partners are involved in cell stress response we think that the helicase could play important role by moduling the cell conditions in response to environment changes.

INSTRUMENT(S): Synapt G2 HDMS

ORGANISM(S): Escherichia Coli (ncbitaxon:562)

SUBMITTER: Algirdas Kaupinis

PROVIDER: MSV000089861 | MassIVE | Sun Jul 10 22:59:00 BST 2022

REPOSITORIES: MassIVE

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Decapping enzyme NUDT12 partners with BLMH for cytoplasmic surveillance of NAD-capped RNAs

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2019-12-25 | GSE138019 | GEO

Landscape of RNA NAD+ capping in Arabidopsis unopened flower bud

Project description:Eukaryotic messenger RNAs (mRNAs) possess a 5’-end N7-methyl guanosine (m7G) cap that promotes their translation and stability. However, it was recently demonstrated that eukaryotic mRNAs can also carry a 5' end nicotinamide adenine dinucleotide (NAD+) cap that promotes mRNA decay mediated by the NAD+ decapping enzyme DXO1. However, the dynamic regulation of NAD+ capping in plant remains unknown. Here, we describe the global landscape of NAD+-capped RNAs in Arabidopsis thaliana, and demonstrate that DXO1 is responsible for removal of these 5’-end modifications and facilitates mRNA degradation in plant transcriptomes. We also reveal that in the absence of DXO1 NAD+-capped mRNAs are unstable and processed into smRNAs. Furthermore, we find that Abscisic Acid (ABA) remodel the landscape of RNA cap epitransciptome, and the mRNA lost their NAD+ cap contribute to their stability under ABA. Overall, our results support a link between ABA response and RNA NAD+ capping.

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Presence of NAD+-capped RNA in human cells: function and removal by the DXO deNADing Protein

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Nudt12 is an mRNA DeNADding Enzyme

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.

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RNA sequencing of DDX23 knockdown by siRNA in ovarian cancer cells

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2021-07-31 | GSE181078 | GEO

The RNA NAD+ cap is widespread in the Arabidopsis transcriptome and probably promotes translation

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

NAD tagSeq, a new method for transcriptome-wide identification and quantification of NAD+-capped RNAs, revealed that NAD+-capped RNAs are produced from a large number of protein-coding genes in Arabidopsis

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.

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