Arabidopsis DXO1 links RNA turnover and chloroplast function independently of its enzymatic activity

Kwaśnik, Aleksandra; Wang, Vivien Ya-Fan; Krzysztoń, Michał; Gozdek, Agnieszka; Zakrzewska‐Placzek, Monika; Stępniak, Karolina; Poznański, Jarosław; Tong, Liang; Kufel, Joanna

doi:10.1093/nar/gkz100

Cited by 29 publications

(47 citation statements)

References 52 publications

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“…SPAAC-NAD-seq will aid future studies on NAD-RNA metabolism and molecular functions. The RNA NAD + cap can be removed by at least two classes of decapping enzymes, Nudix family proteins present in both prokaryotes and eukaryotes (8,(33)(34)(35) and DXO proteins found only in eukaryotes (9,33,36,37). NAD-RNAs transfected into human cells and Arabidopsis protoplasts undergo DXO-dependent degradation (9,37).…”

Section: Discussionmentioning

confidence: 99%

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SPAAC-NAD-seq, a sensitive and accurate method to profile NAD ⁺ -capped transcripts

Flynn

Zhang

et al. 2021

Proc. Natl. Acad. Sci. U.S.A.

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Nicotinamide adenine diphosphate (NAD+) is a novel messenger RNA 5′ cap in Escherichia coli, yeast, mammals, and Arabidopsis. Transcriptome-wide identification of NAD+-capped RNAs (NAD-RNAs) was accomplished through NAD captureSeq, which combines chemoenzymatic RNA enrichment with high-throughput sequencing. NAD-RNAs are enzymatically converted to alkyne-RNAs that are then biotinylated using a copper-catalyzed azide–alkyne cycloaddition (CuAAC) reaction. Originally applied to E. coli RNA, which lacks the m7G cap, NAD captureSeq was then applied to eukaryotes without extensive verification of its specificity for NAD-RNAs vs. m7G-capped RNAs (m7G-RNAs). In addition, the Cu2+ ion in the CuAAC reaction causes RNA fragmentation, leading to greatly reduced yield and loss of full-length sequence information. We developed an NAD-RNA capture scheme utilizing the copper-free, strain-promoted azide–alkyne cycloaddition reaction (SPAAC). We examined the specificity of CuAAC and SPAAC reactions toward NAD-RNAs and m7G-RNAs and found that both prefer the former, but also act on the latter. We demonstrated that SPAAC-NAD sequencing (SPAAC-NAD-seq), when combined with immunodepletion of m7G-RNAs, enables NAD-RNA identification with accuracy and sensitivity, leading to the discovery of new NAD-RNA profiles in Arabidopsis. Furthermore, SPAAC-NAD-seq retained full-length sequence information. Therefore, SPAAC-NAD-seq would enable specific and efficient discovery of NAD-RNAs in prokaryotes and, when combined with m7G-RNA depletion, in eukaryotes.

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Section: Discussionmentioning

confidence: 99%

“…NAD-RNAs transfected into human cells and Arabidopsis protoplasts undergo DXO-dependent degradation (9,37). In an Arabidopsis dxo mutant, NAD + -capped transcripts are channeled to RNA silencing for degradation (32,36). NAD captureSeq was performed in yeast, human, and Arabidopsis dxo mutants to identify the in vivo targets of DXO (9,13,32).…”

Section: Discussionmentioning

confidence: 99%

SPAAC-NAD-seq, a sensitive and accurate method to profile NAD ⁺ -capped transcripts

Flynn

Zhang

et al. 2021

Proc. Natl. Acad. Sci. U.S.A.

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“…This human enzyme uses a decapping chemistry entirely different from the two known classes of NAD decapping enzymes, as it converts NAD-RNA into 5'-ADP-ribose-RNA, liberating nicotinamide ( Figure 6). In contrast, the prokaryotic decapping enzymes NudC and BsRppH [3,8,32] and the putative eukaryotic NAD-decapping enzyme Npy1 [51] hydrolyse the pyrophosphate bond within NAD, while DXO enzymes take off the entire NAD cap [10,18,21]. Interestingly, Npy1p, DXO1 and CD38 are differently localized in the cell.…”

Section: Discussionmentioning

confidence: 99%

“…Moreover, the mammalian and fungal DXO1/Rai1 enzymes were described to remove-in addition to the eukaryotic m 7 G-cap-the NAD modification. In contrast to the Nudix hydrolases, the DXO enzymes cut off the complete NAD cap including the adenosine, generating a 5'-P-RNA shortened by one nucleotide [10,21].…”

Section: Introductionmentioning

confidence: 99%

A Novel NAD-RNA Decapping Pathway Discovered by Synthetic Light-Up NAD-RNAs

et al. 2020

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The complexity of the transcriptome is governed by the intricate interplay of transcription, RNA processing, translocation, and decay. In eukaryotes, the removal of the 5’-RNA cap is essential for the initiation of RNA degradation. In addition to the canonical 5’-N7-methyl guanosine cap in eukaryotes, the ubiquitous redox cofactor nicotinamide adenine dinucleotide (NAD) was identified as a new 5’-RNA cap structure in prokaryotic and eukaryotic organisms. So far, two classes of NAD-RNA decapping enzymes have been identified, namely Nudix enzymes that liberate nicotinamide mononucleotide (NMN) and DXO-enzymes that remove the entire NAD cap. Herein, we introduce 8-(furan-2-yl)-substituted NAD-capped-RNA (FurNAD-RNA) as a new research tool for the identification and characterization of novel NAD-RNA decapping enzymes. These compounds are found to be suitable for various enzymatic reactions that result in the release of a fluorescence quencher, either nicotinamide (NAM) or nicotinamide mononucleotide (NMN), from the RNA which causes a fluorescence turn-on. FurNAD-RNAs allow for real-time quantification of decapping activity, parallelization, high-throughput screening and identification of novel decapping enzymes in vitro. Using FurNAD-RNAs, we discovered that the eukaryotic glycohydrolase CD38 processes NAD-capped RNA in vitro into ADP-ribose-modified-RNA and nicotinamide and therefore might act as a decapping enzyme in vivo. The existence of multiple pathways suggests that the decapping of NAD-RNA is an important and regulated process in eukaryotes.

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“…The NAD + ‐capped mRNAs are polyadenylated, spliced, and found in polysomes (Y. Wang et al, 2019). Proteins responsible for removing NAD + are also encoded in plant genomes, but may not function in this capacity (Kwasnik et al, 2019; Pan et al, 2019). For all these covalent mRNA modifications, their immediate impact on translation is not well defined but m 6 A is known to lower or boost mRNA stability (S. J. Anderson et al, 2018; Shen et al, 2016).…”

Section: Rna Featuresmentioning

confidence: 99%

Translational gene regulation in plants: A green new deal

2020

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The molecular machinery for protein synthesis is profoundly similar between plants and other eukaryotes. Mechanisms of translational gene regulation are embedded into the broader network of RNA-level processes including RNA quality control and RNA turnover. However, over eons of their separate history, plants acquired new components, dropped others, and generally evolved an alternate way of making the parts list of protein synthesis work. Research over the past 5 years has unveiled how plants utilize translational control to defend themselves against viruses, regulate translation in response to metabolites, and reversibly adjust translation to a wide variety of environmental parameters. Moreover, during seed and pollen development plants make use of RNA granules and other translational controls to underpin developmental transitions between quiescent and metabolically active stages. The economics of resource allocation over the daily light-dark cycle also include controls over cellular protein synthesis. Important new insights into translational control on cytosolic ribosomes continue to emerge from studies of translational control mechanisms in viruses. Finally, sketches of coherent signaling pathways that connect external stimuli with a translational response are emerging, anchored in part around TOR and GCN2 kinase signaling networks. These again reveal some mechanisms that are familiar and others that are different from other eukaryotes, motivating deeper studies on translational control in plants.

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Arabidopsis DXO1 links RNA turnover and chloroplast function independently of its enzymatic activity

Cited by 29 publications

References 52 publications

SPAAC-NAD-seq, a sensitive and accurate method to profile NAD ⁺ -capped transcripts

SPAAC-NAD-seq, a sensitive and accurate method to profile NAD ⁺ -capped transcripts

A Novel NAD-RNA Decapping Pathway Discovered by Synthetic Light-Up NAD-RNAs

Translational gene regulation in plants: A green new deal

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Arabidopsis DXO1 links RNA turnover and chloroplast function independently of its enzymatic activity

Cited by 29 publications

References 52 publications

SPAAC-NAD-seq, a sensitive and accurate method to profile NAD + -capped transcripts

SPAAC-NAD-seq, a sensitive and accurate method to profile NAD + -capped transcripts

A Novel NAD-RNA Decapping Pathway Discovered by Synthetic Light-Up NAD-RNAs

Translational gene regulation in plants: A green new deal

Contact Info

Product

Resources

About

SPAAC-NAD-seq, a sensitive and accurate method to profile NAD ⁺ -capped transcripts

SPAAC-NAD-seq, a sensitive and accurate method to profile NAD ⁺ -capped transcripts