Cellular metabolites have emerged as noncanonical RNA caps. Despite its early discovery as an RNA cap, the dephospho-CoA (dpCoA) cap remains largely uncharacterized because of a lack of detection technologies. Here we use biochemical and structural analysis to identify Arabidopsis NUDT11 as a specific decapping enzyme toward dpCoA-RNA.
Cellular metabolites have emerged as noncanonical RNA caps. Despite its early discovery as an RNA cap, the dephospho-CoA cap remains largely uncharacterized because of a lack of detection technologies.
Here we use biochemical and structural analysis to identifyNUDT11 as a specific decapping enzyme toward dpCoA-RNA. Leveraging this specificity, we develop biochemical and transcriptomic methods to quantify and profile dpCoA-RNA across the genome, revealing that dpCoA-RNAs exist across species and exhibit tissue-specific and/or condition-specific variations. InG-capped RNAs in abundance and are associated with translating ribosomes. We further demonstrate that an in vitro transcribed dpCoA-RNA is translated in human cells. This study uncovers a dynamic dpCoA cap that may potentially influence gene expression and establishes a toolkit for future investigations.Fig. 1: Detection of dpCoA-RNA in various organisms by QQQ-LC–MS.Fig. 3: Quantification of dpCoA-RNA in various organisms using dpCoA-TLC.The raw sequence data generated in this study were deposited to the NCBI Gene Expression Omnibus database under accession codeLi, X., Egervari, G., Wang, Y., Berger, S. L. & Lu, Z. Regulation of chromatin and gene expression by metabolic enzymes and metabolites.Chen, Y. G., Kowtoniuk, W. E., Agarwal, I., Shen, Y. & Liu, D. R. LC/MS analysis of cellular RNA reveals NAD-linked RNA.Kowtoniuk, W. E., Shen, Y., Heemstra, J. M., Agarwal, I. & Liu, D. R. A chemical screen for biological small molecule–RNA conjugates reveals CoA-linked RNA.Cahová, H., Winz, M.-L., Höfer, K., Nübel, G. & Jäschke, A. NAD captureSeq indicates NAD as a bacterial cap for a subset of regulatory RNAs.Sharma, S., Yang, J., Favate, J., Shah, P. & Kiledjian, M. NADcapPro and circNC: methods for accurate profiling of NAD and non-canonical RNA caps in eukaryotes.Google ScholarGoogle ScholarGasmi, L. & Mclennan, A. G. The mouse Nudt7 gene encodes a peroxisomal nudix hydrolase specific for coenzyme A and its derivatives.Meijer, H. A., Dictus, W. J. A. G., Keuning, E. D. & Thomas, A. A. M. Translational control of theGoogle ScholarMickutė, M. et al. Interplay between bacterial 5′-NAD-RNA decapping hydrolase NudC and DEAD-box RNA helicase CsdA in stress responses.Luciano, D. J., Levenson-Palmer, R. & Belasco, J. G. Stresses that raise Np4A levels induce protective nucleoside tetraphosphate capping of bacterial RNA.Khan, I., Sohail, Zaman, S., Li, G. & Fu, M. Adaptive responses of plants to light stress: mechanisms of photoprotection and acclimation. A review.Google ScholarNiedzwiecka, A. et al. Biophysical studies of eIF4E cap-binding protein: recognition of mRNA 5′ cap structure and synthetic fragments of eIF4G and 4E-BP1 proteins.Chen, Y., Chen, L., Lun, A. T. L., Baldoni, P. L. & Smyth, G. K. edgeR v4: powerful differential analysis of sequencing data with expanded functionality and improved support for small counts and larger datasets.We would like to express our gratitude to I. O. Vvedenskaya from Rutgers University for the suggestions on dpCoA-CapZyme-seq library construction. We thank H. Wu and J. Liu from Peking University for supplying mouse and human cell materials. We thank S. Huang from the Isotope Laboratory at Peking University for assistance with experiments involving radioactive reagents. We thank the Core Facilities of the School of Life Sciences and National Center for Protein Sciences at Peking University for assistance with protein purification by size-exclusion chromatography. We thank Y. Xia from Hong Kong Baptist University for sharing the expression plasmids of SpRai1 and AtDXO. We thank H. Wen for assistance in predicting the key amino acid sites of AtNUDT11. We thank C. Yi and J. Liu for comments on the manuscript. Research in the X.C. laboratory is supported by the State Key Laboratory for Gene Function and Modulation Research, Peking-Tsinghua Joint Center for Life Sciences and Beijing Advanced Center of RNA Biology. This work was supported by National Natural Science Foundation of China and Beijing Natural Science Foundation to H.H., National Key R&D Program of China to X.C. and US National Institutes of Health R35GM118093 to L.T. This work is based in part on research conducted at the Northeastern Collaborative Access Team beamlines, which are funded by the National Institute of General Medical Sciences from the National Institutes of Health . This research used resources of the Advanced Photon Source, a US Department of Energy Office of Science User Facility operated for the DOE Office of Science by Argonne National Laboratory under contract no. DE-AC02-06CH11357.Beijing Advanced Center of RNA Biology , State Key Laboratory for Gene Function and Modulation Research, Peking-Tsinghua Joint Center for Life Sciences, School of Life Sciences, Peking University, Beijing, China Hao Hu, Qiyue Zhang, Han Wang, Yiwen Guo, Yuze Bai, Qianyu Wang, Jiayi Zhao, Huiyuan Lin & Xuemei ChenInterdisciplinary Science Center, State Key Laboratory of Integrated Management of Pest Insects and Rodents, Institute of Zoology, Chinese Academy of Sciences, Beijing, ChinaCollege of Life Sciences, Guangdong Provincial Key Laboratory for the Development Biology and Environmental Adaptation of Agricultural Organisms, South China Agricultural University, Guangzhou, Guangdong, China X.C. and H.H. conceptualized the project and designed the experiments. H.H., Q.Z., Y.G., Y.B., Q.W., Z.L., J.Z. and H.L. performed the experiments. X.M. and H.W. developed the bioinformatics pipeline of dpCoA-CapZyme-seq. L.T. and H.C. solved the crystal structure of AtNUDT11. C.Y. provided assistance in dpCoA-CapZyme-seq data analysis. H.H., Q.Z., Y.G., H.C., Z.L., L.T. and X.C. interpreted the results and wrote the manuscript. X.C. and H.H. supervised the project.thanks Katharina Höfer, Yiji Xia and the other, anonymous, reviewer for their contribution to the peer review of this work. , An in vitro transcribed 31-nucleotide dpCoA-RNA was incubated with or without different decapping enzymes followed by electrophoresis in a 15% polyacrylamide APB gel. The positions of the dpCoA-RNA and the cleavage product are indicated. The smaller RNA fragments represent further degradation by mDXO and AtDXO, which have 5′ to 3′ exonuclease activityNudix hydrolases, as well as human and yeast Nudix hydrolases with CoA pyrophosphohydrolase activity, and bacterial NudC. The scale bar for branch lengths represents 0.2 amino acid substitutions per site.NUDTs in 2 h at 37 °C, with buffer alone as the control. The dpCoA peak is marked. The large peak present in each chromatogram is a solvent peak. The bar plot to the right shows the amount of dpCoA remaining after incubation for the specified time periods. The peak areas of dpCoA were measured, and the relative amount of dpCoA in each treatment was normalized to the peak area of the buffer-treated sample. Three biological replicates were performed, and error bars represent the mean ± s.e.m., HPLC chromatograms showing each of four metabolites treated with various AtNUDTs for 2 h at 37 °C.. The peak areas corresponding to each compound were integrated, and the relative amounts in each treatment were normalized to the peak area of the buffer control. Three independent biological replicates were conducted. Error bars represent the mean ± s.e.m., Partial amino acid sequences surrounding the Nudix motifs of AtNUDT11, AtNUDT15, and AtNUDT22, along with other Nudix hydrolases exhibiting dpCoA pyrophosphohydrolase activity across various organisms, were aligned. The UPF0035 motif and the Nudix motif are indicated. The red triangles denote the amino acids mutated to generate catalytically inactive enzymes., An in vitro transcribed dpCoA-RNA was treated with or without AtNUDTs and analyzed using a 15% polyacrylamide APB gel. The positions of the dpCoA-RNA and the cleavage product are indicated. Data are representative of three independent experiments with similar results.A-RNA, as indicated, was incubated with or without AtNUDT11, AtNUDT15 or AtNUDT22. The reaction products were analyzed in 15% polyacrylamide APB gels. The positions of the capped RNA are marked with arrows. The gels were stained with the SYBRGpppA treated with various decapping enzymes, with buffer-only reactions serving as mock controls. Arrows indicate the compounds represented by the corresponding peaks.. The error bars indicate the mean ± s.e.m. calculated from three independent experiments. Statistical significance was determined using a two-tailed Student′sG-RNA following enzymatic decapping. Reaction products were resolved in a 15% polyacrylamide APB gel and transferred to a nylon Nmembrane. RNA was hybridized with a biotin-labeled probe, detected using streptavidin-horseradish peroxidase, and visualized with a chemiluminescent nucleic acid detection module. Positive controls were dpCoA-RNA treated with AtNUDT11 and mG-RNA treated with hDCP2. Black arrows indicate the positions of the original RNA substrates and blue arrows indicate the positions of p-RNA. Data are representative of two independent experiments with similar results., Decapping kinetics of dpCoA-RNA in vitro. The amount of dpCoA-RNA remaining at each time point was measured and plotted based on data from three independent experiments, with the error bars representing the mean ± s.e.m.represent buffers containing the corresponding metal ion. All decapping reactions were incubated at 37 °C, except for one reaction incubated at 4 °C as indicated. Data are representative of three independent experiments with similar results., A time course of decapping assays using a mixture of equal amounts of dpCoA-RNA and NAD-RNA with AtNUDT11, AtNUDT15 and AtNUDT22. RNAs were separated in 15% polyacrylamide APB gels following the reactions. The remaining capped RNAs at each time point were quantified using ImageJ. Data from three independent experiments were plotted on the right, with error bars representing the mean ± s.e.m., The decapping kinetics of AtNUDT11 on dpCoA-RNA assessed in the presence of 500-fold excess of dpCoA. The RNA was analyzed using a 15% polyacrylamide APB gel to determine the amount of the remaining dpCoA-RNA. The results were derived from three independent experiments, with error bars representing the mean ± s.e.m. , HPLC chromatograms showing complete hydrolysis of dpCoA by AtNUDT11, followed by the production of new dpCoA with the addition of PPAT and ATP., TLC analysis to evaluate of the effectiveness of the NAP-10 column in removing free dpCoA from total RNA for dpCoA-TLC assays. Total RNA was incubated with 10 nmol or 100 nmol of -dpCoA cap signal was visualized with a Typhoon phosphorimager. Quantification of the detected signals is presented as a bar plot in Fig.P]-dpCoA cap signal was visualized with a Typhoon phosphorimager. Quantification of the detected signals is presented as a bar plot in Fig., Detection of dpCoA-RNA in poly RNA from yeast grown under two conditions, normal and sugar stress, using dpCoA-TLC. The -dpCoA cap signal was visualized with a Typhoon phosphorimager. Quantification of the detected signals is presented as bar plots in Fig., Bar charts showing the quantification of dpCoA-RNA in total RNA from human cells and yeast . The amounts were determined from a calibration curve generated from a series of dpCoA standards. Error bars represent the mean ± s.e.m. from three independent replicates. The original TLC images corresponding to the bar plots are shown in, mouse, human cells, and yeast. Free dpCoA was quantified using QQQ-LC-MS, and the concentrations were determined based on a calibration curve generated from serially diluted dpCoA standards. An internal standard “adenosine-1’-13C” was used to correct for sample handling variability. Error bars represent the mean ± s.e.m. from three independent biological replicates. , The detailed workflow for dpCoA-CapZyme-seq library construction. CIP was first used to remove most of 5′ p-RNA from poly RNA. dpCoA-RNAs were then decapped by AtNUDT11, resulting in RNAs with 5′-monophosphate ends, which were then ligated to a 5′ adaptor oligonucleotide. Reverse transcription was performed using random primers containing a known sequence handle, enabling the construction of a library. Cartoons of the objects were created withtotal RNA, confirming that AtNUDT11 exhibits no RNA degradation activity. RNase T1 was used as a positive control to indicate RNA degradation. Data are from one independent experiment.genome into 10-bp bins, with the number of 5′ end reads in each bin counted. Bins exhibiting significantly enriched read counts , while other RNA types lacking the dpCoA modification migrate through the APM layer., APM gel analysis with in vitro transcribed RNAs with various caps, demonstrating that only dpCoA-RNA was retained within the APM layer. In contrast, other RNA species, including ppp-RNA, NAD-RNA, mas dpCoA-RNA. RNA recovered from the APM-containing region were excised and reacted with maleimide-PEG2-biotin. The in vitro transcribed dpCoA-RNA without APM gel electrophoresis was included as a positive control. The biotin-conjugated products were resolved in a 15% polyacrylamide gel, transferred to a nylon N⁺ membrane, and detected via streptavidin–horseradish peroxidase using a chemiluminescent nucleic acid detection kit. Data are representative of three independent experiments with similar results., Structural features of dpCoA-RNA-producing genes, including gene length, exon length, intron length, exon number, and intron number, were assessed using a permutation test, following the same method as described in Fig., Seqlogo analysis of the sequences spanning the −10 to +6 region surrounding the annotated TSS. Two groups of randomly selected RNAs in addition to the one group shown in Fig.Springer Nature or its licensor holds exclusive rights to this article under a publishing agreement with the author or other rightsholder; author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law.
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