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Proceedings of the National Academy of... Sep 2023In the final step of cytokinin biosynthesis, the main pathway is the elimination of a ribose-phosphate moiety from the cytokinin nucleotide precursor by...
In the final step of cytokinin biosynthesis, the main pathway is the elimination of a ribose-phosphate moiety from the cytokinin nucleotide precursor by phosphoribohydrolase, an enzyme encoded by a gene named LONELY GUY (LOG). This reaction accounts for most of the cytokinin supply needed for regulating plant growth and development. In contrast, the LOG-independent pathway, in which dephosphorylation and deribosylation sequentially occur, is also thought to play a role in cytokinin biosynthesis, but the gene entity and physiological contribution have been elusive. In this study, we profiled the phytohormone content of chromosome segment substitution lines of and searched for genes affecting the endogenous levels of cytokinin ribosides by quantitative trait loci analysis. Our approach identified a gene encoding an enzyme that catalyzes the deribosylation of cytokinin nucleoside precursors and other purine nucleosides. The cytokinin/purine riboside nucleosidase 1 (CPN1) we identified is a cell wall-localized protein. Loss-of-function mutations () were created by inserting a -retrotransposon that altered the cytokinin composition in seedling shoots and leaf apoplastic fluid. The mutation also abolished cytokinin riboside nucleosidase activity in leaf extracts and attenuated the -zeatin riboside-responsive expression of cytokinin marker genes. Grain yield of the mutants declined due to altered panicle morphology under field-grown conditions. These results suggest that the cell wall-localized LOG-independent cytokinin activating pathway catalyzed by CPN1 plays a role in cytokinin control of rice growth. Our finding broadens our spatial perspective of the cytokinin metabolic system.
Topics: Oryza; Cytokinins; Purine Nucleosides; N-Glycosyl Hydrolases; Nucleosides; Cell Wall
PubMed: 37639600
DOI: 10.1073/pnas.2217708120 -
Experimental Physiology Nov 2023
Topics: Adenosine; Receptors, Purinergic P1
PubMed: 37720963
DOI: 10.1113/EP091467 -
Microbiology Spectrum Aug 2022Toyocamycin (TM) is an adenosine-analog antibiotic isolated from Streptomyces toyocaensis. It inhibits Candida albicans, several plant fungal pathogens, and human cells,...
Toyocamycin (TM) is an adenosine-analog antibiotic isolated from Streptomyces toyocaensis. It inhibits Candida albicans, several plant fungal pathogens, and human cells, but many fungi, including Saccharomyces cerevisiae, are much less susceptible to TM. Aiming to clarify why TM and its analogs tubercidin and 5-iodotubercidin are active against C. albicans but not S. cerevisiae, this study focused on the absence of purine nucleoside transport activity from S. cerevisiae. When the concentrative nucleoside transporter (CNT) of C. albicans was expressed in S. cerevisiae, the recombinant strain became sensitive to TM and its analogs. The expression of C. albicans purine nucleoside permease in S. cerevisiae did not result in sensitivity to TM. Clustered regularly interspaced short palindromic repeat-mediated disruption of CNT was performed in C. albicans. The CNTΔ strain of C. albicans became insensitive to TM and its analogs. These data suggest that the toxicity of TM and its analogs toward C. albicans results from their transport via CNT. Interestingly, S. cerevisiae also became sensitive to TM and its analogs if human CNT3 was introduced into cells. These findings enhance our understanding of the mechanisms of action of adenosine analogs toward pathogens and human cells. We investigated the mechanism of toxicity of TM and its analogs to C. albicans. Inspired by the effect of the copresence of TM and purine nucleosides on cell growth of C. albicans, we investigated the involvement of CNT in the toxicity mechanism by expressing CNT of C. albicans (CaCNT) in S. cerevisiae and deleting CaCNT in C. albicans. Our examinations clearly demonstrated that CaCNT is responsible for the toxicity of TM to C. albicans. S. cerevisiae expressing the human ortholog of CaCNT also became sensitive to TM and its analogs, and the order of effects of the TM analogs was a little different between CaCNT- and hCNT3-expressing S. cerevisiae. These findings are beneficial for an understanding of the mechanisms of action of adenosine analogs toward pathogens and human cells and also the development of new antifungal drugs.
Topics: Adenosine; Candida albicans; Humans; Nucleoside Transport Proteins; Purine Nucleosides; Saccharomyces cerevisiae; Toyocamycin
PubMed: 35913167
DOI: 10.1128/spectrum.01138-22 -
The Journal of Biological Chemistry Nov 2021Tandem mass spectrometry (MS/MS) is an accurate tool to assess modified ribonucleosides and their dynamics in mammalian cells. However, MS/MS quantification of lowly...
Tandem mass spectrometry (MS/MS) is an accurate tool to assess modified ribonucleosides and their dynamics in mammalian cells. However, MS/MS quantification of lowly abundant modifications in non-ribosomal RNAs is unreliable, and the dynamic features of various modifications are poorly understood. Here, we developed a C labeling approach, called C-dynamods, to quantify the turnover of base modifications in newly transcribed RNA. This turnover-based approach helped to resolve mRNA from ncRNA modifications in purified RNA or free ribonucleoside samples and showed the distinct kinetics of the N6-methyladenosine (mA) versus 7-methylguanosine (mG) modification in polyA+-purified RNA. We uncovered that N6,N6-dimethyladenosine (mA) exhibits distinct turnover in small RNAs and free ribonucleosides when compared to known mA-modified large rRNAs. Finally, combined measurements of turnover and abundance of these modifications informed on the transcriptional versus posttranscriptional sensitivity of modified ncRNAs and mRNAs, respectively, to stress conditions. Thus, C-dynamods enables studies of the origin of modified RNAs at steady-state and subsequent dynamics under nonstationary conditions. These results open new directions to probe the presence and biological regulation of modifications in particular RNAs.
Topics: Adenosine; Carbon Isotopes; Guanosine; Isotope Labeling; RNA; RNA Processing, Post-Transcriptional; Tandem Mass Spectrometry
PubMed: 34634303
DOI: 10.1016/j.jbc.2021.101294 -
Free Radical Biology & Medicine Sep 2023There is accumulating evidence that pro-inflammatory features are inherent to mitochondrial DNA and oxidized DNA species. 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodGuo)...
There is accumulating evidence that pro-inflammatory features are inherent to mitochondrial DNA and oxidized DNA species. 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodGuo) is the most frequently studied oxidatively generated lesion. Modified DNA reaches the circulation upon cell apoptosis, necrosis or neutrophil extracellular trap (NET) formation. Standard chromatography-based techniques for the assessment of 8-oxodGuo imply degradation of DNA to a single base level, thus precluding the attribution to a nuclear or mitochondrial origin. We therefore aimed to establish a protocol for the concomitant assessment of oxidized mitochondrial and nuclear DNA from human plasma samples. We applied immunoprecipitation (IP) for 8-oxodGuo to separate oxidized from non-oxidized DNA species and subsequent quantitative polymerase chain reaction (qPCR) to assign them to their subcellular source. The IP procedure failed when applied directly to plasma samples, i.e. isotype control precipitated similar amounts of DNA as the specific 8-oxodGuo antibody. In contrast, DNA isolation from plasma prior to the IP process provided assay specificity with little impact on DNA oxidation status. We further optimized sensitivity and efficiency of qPCR analysis by reducing amplicon length and targeting repetitive nuclear DNA elements. When the established protocol was applied to plasma samples of abdominal aortic aneurysm (AAA) patients and control subjects, the AAA cohort displayed significantly elevated circulating non-oxidized and total nuclear DNA and a trend for increased levels of oxidized mitochondrial DNA. An enrichment of mitochondrial versus nuclear DNA within the oxidized DNA fraction was seen for AAA patients. Regarding the potential source of circulating DNA, we observed a significant correlation of markers of neutrophil activation and NET formation with nuclear DNA, independent of oxidation status. Thus, the established method provides a tool to detect and distinguish the release of oxidized nuclear and mitochondrial DNA in human plasma and offers a refined biomarker to monitor disease conditions of pro-inflammatory cell and tissue destruction.
Topics: Humans; 8-Hydroxy-2'-Deoxyguanosine; Deoxyguanosine; DNA, Mitochondrial; Oxidation-Reduction; Aortic Aneurysm, Abdominal
PubMed: 37353175
DOI: 10.1016/j.freeradbiomed.2023.06.014 -
Communications Biology Apr 2022The global dietary supplement market is valued at over USD 100 billion. One popular dietary supplement, S-adenosylmethionine, is marketed to improve joints, liver health...
The global dietary supplement market is valued at over USD 100 billion. One popular dietary supplement, S-adenosylmethionine, is marketed to improve joints, liver health and emotional well-being in the US since 1999, and has been a prescription drug in Europe to treat depression and arthritis since 1975, but recent studies questioned its efficacy. In our body, S-adenosylmethionine is critical for the methylation of nucleic acids, proteins and many other targets. The marketing of SAM implies that more S-adenosylmethionine is better since it would stimulate methylations and improve health. Previously, we have shown that methylation reactions regulate biological rhythms in many organisms. Here, using biological rhythms to assess the effects of exogenous S-adenosylmethionine, we reveal that excess S-adenosylmethionine disrupts rhythms and, rather than promoting methylation, is catabolized to adenine and methylthioadenosine, toxic methylation inhibitors. These findings further our understanding of methyl metabolism and question the safety of S-adenosylmethionine as a supplement.
Topics: Adenine; Dietary Supplements; Liver; Methylation; S-Adenosylmethionine
PubMed: 35383287
DOI: 10.1038/s42003-022-03280-5 -
International Journal of Molecular... Jan 2023The guanine base in nucleic acids is, among the other bases, the most susceptible to being converted into 8-Oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG) when exposed to... (Review)
Review
The guanine base in nucleic acids is, among the other bases, the most susceptible to being converted into 8-Oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG) when exposed to reactive oxygen species. In double-helix DNA, 8-oxodG can pair with adenine; hence, it may cause a G > T (C > A) mutation; it is frequently referred to as a form of DNA damage and promptly corrected by DNA repair mechanisms. Moreover, 8-oxodG has recently been redefined as an epigenetic factor that impacts transcriptional regulatory elements and other epigenetic modifications. It has been proposed that 8-oxodG exerts epigenetic control through interplay with the G-quadruplex (G4), a non-canonical DNA structure, in transcription regulatory regions. In this review, we focused on the epigenetic roles of 8-oxodG and the G4 and explored their interplay at the genomic level.
Topics: 8-Hydroxy-2'-Deoxyguanosine; Deoxyguanosine; DNA Damage; DNA Repair; DNA
PubMed: 36768357
DOI: 10.3390/ijms24032031 -
Making Changes: N-Methyladenosine-Mediated Decay Drives the Endothelial-to-Hematopoietic Transition.Biochemistry Nov 2017Zhang et al. (2017) report critical functions of mA during the endothelial-to-haematopoietic transition in zebrafish embryogenesis. mA affects Notch signaling, likely... (Review)
Review
Zhang et al. (2017) report critical functions of mA during the endothelial-to-haematopoietic transition in zebrafish embryogenesis. mA affects Notch signaling, likely though mA-dependent and YTHDF2-mediated mRNA decay.
Topics: Adenosine; Animals; Endothelial Cells; Hematopoietic Stem Cells; Humans; Receptors, Notch
PubMed: 29112411
DOI: 10.1021/acs.biochem.7b01077 -
Molecules (Basel, Switzerland) Apr 2020Nucleoside analogues have proven to be highly successful chemotherapeutic agents in the treatment of a wide variety of cancers. Several such compounds, including... (Review)
Review
Nucleoside analogues have proven to be highly successful chemotherapeutic agents in the treatment of a wide variety of cancers. Several such compounds, including gemcitabine and cytarabine, are the go-to option in first-line treatments. However, these materials do have limitations and the development of next generation compounds remains a topic of significant interest and necessity. Herein, we discuss recent advances in the chemical synthesis and biological evaluation of nucleoside analogues as potential anticancer agents. Focus is paid to 4'-heteroatom substitution of the furanose oxygen, 2'-, 3'-, 4'- and 5'-position ring modifications and the development of new prodrug strategies for these materials.
Topics: Adenosine; Animals; Antineoplastic Agents; Cell Line, Tumor; Drug Design; Drug Screening Assays, Antitumor; Furans; Humans; K562 Cells; Mice; Molecular Structure; Nucleosides; Oxygen; Prodrugs; Purine Nucleosides; Pyrimidinones; Thionucleosides; Vitamin E
PubMed: 32354007
DOI: 10.3390/molecules25092050 -
Nature Communications Jun 2021Methylation is a prevalent post-transcriptional modification encountered in coding and non-coding RNA. For RNA methylation, cells use methyltransferases and small...
Methylation is a prevalent post-transcriptional modification encountered in coding and non-coding RNA. For RNA methylation, cells use methyltransferases and small organic substances as methyl-group donors, such as S-adenosylmethionine (SAM). SAM and other nucleotide-derived cofactors are viewed as evolutionary leftovers from an RNA world, in which riboswitches have regulated, and ribozymes have catalyzed essential metabolic reactions. Here, we disclose the thus far unrecognized direct link between a present-day riboswitch and its inherent reactivity for site-specific methylation. The key is O-methyl pre-queuosine (mpreQ), a potentially prebiotic nucleobase which is recognized by the native aptamer of a preQ class I riboswitch. Upon binding, the transfer of the ligand's methyl group to a specific cytidine occurs, installing 3-methylcytidine (mC) in the RNA pocket under release of pre-queuosine (preQ). Our finding suggests that nucleic acid-mediated methylation is an ancient mechanism that has offered an early path for RNA epigenetics prior to the evolution of protein methyltransferases. Furthermore, our findings may pave the way for the development of riboswitch-descending methylation tools based on rational design as a powerful alternative to in vitro selection approaches.
Topics: Aptamers, Nucleotide; Base Sequence; Kinetics; Methylation; Molecular Structure; Nucleic Acid Conformation; Nucleoside Q; RNA; Riboswitch; S-Adenosylmethionine
PubMed: 34162884
DOI: 10.1038/s41467-021-24193-7