Advances in the investigation of N6-isopentenyl adenosine i6A RNA modification.

Prenylation (isopentenylation), a key post-transcriptional modification with a hydrophobic prenyl group onto the biomacromolecules such as RNA and proteins, influences their localization and function. Prenyltransferases mediate this process, while cytokinin oxidases degrade the prenylated adenosine in plants. This review summarizes current progress in detecting prenylation modifications in RNA across species and their effects on protein synthesis. Advanced methods have been developed to label and study these modifications in vitro and in vivo, despite challenges posed by the inert chemical properties of prenyl groups. Continued advancements in bioorthogonal chemistry promise new tools for understanding the precise biological functions of prenylated RNA modifications and other related proteins.

The Transcriptome-Wide Mapping of 2-Methylthio-N6-isopentenyladenosine at Single-Base Resolution.

Hundreds of modified bases have been identified and enzymatically modified to transfer RNAs (tRNAs) to regulate RNA function in various organisms. 2-Methylthio-N6-isopentenyladenosine (ms2i6A), a hypermodified base found at tRNA position 37, exists in both prokaryotes and eukaryotes. ms2i6A is traditionally identified by separating and digesting each tRNA from total RNA using RNA mass spectrometry. A transcriptome-wide and single-base resolution method that enables absolute mapping of ms2i6A along with analysis of its distribution in different RNAs is lacking. Here, through chemoselective methylthio group bioconjugation, we introduce a new approach (redox activated chemical tagging sequencing, ReACT-seq) to detect ms2i6A transcriptome-wide at single-base resolution. Using the chemoselectivity between the methylthio group and oxaziridine group, ms2i6A is bio-orthogonally tagged with an azide group without interference of canonical nucleotides, advancing enrichment of methylthio group modified RNAs prior to sequencing. ReACT-seq was demonstrated on nine known tRNAs and proved to be highly accurate, and the reverse transcription stop (RT-stop) character enables ReACT-seq detection at single-base resolution. In addition, ReACT-seq identified that the modification of ms2i6A is conservative and may not exist in other RNAs.

Identification of 2-Methylthio-methylenethio-N6 -(cis-4-hydroxyisopentenyl)adenosine (msms2 io6 A37 ) as a Novel Modification at Adenosine 37 of tRNAs from Salmonella typhimurium.

Post-transcriptional modifications of tRNA nucleotide are important determinants in folding, structure and function. We have successfully identified and characterized a new modified base named 2-methylthio-methylenethio-N6 -(cis-4-hydroxyisopentenyl)adenosine, which is present at position 37 in some tRNAs. We also showed that this new modified adenosine is derived from the known 2-methylthio-methylenethio-N6 -(isopentenyl)adenosine nucleoside by a catalytic cycle of the tRNA-diiron monooxygenase, MiaE, present in Salmonella typhimurium.

Posttranscriptional modifications at the 37th position in the anticodon stem-loop of tRNA: structural insights from MD simulations.

Transfer RNA (tRNA) is the most diversely modified RNA. Although the strictly conserved purine position 37 in the anticodon stem-loop undergoes modifications that are phylogenetically distributed, we do not yet fully understand the roles of these modifications. Therefore, molecular dynamics simulations are used to provide molecular-level details for how such modifications impact the structure and function of tRNA. A focus is placed on three hypermodified base families that include the parent i6A, t6A, and yW modifications, as well as derivatives. Our data reveal that the hypermodifications exhibit significant conformational flexibility in tRNA, which can be modulated by additional chemical functionalization. Although the overall structure of the tRNA anticodon stem remains intact regardless of the modification considered, the anticodon loop must rearrange to accommodate the bulky, dynamic hypermodifications, which includes changes in the nucleotide glycosidic and backbone conformations, and enhanced or completely new nucleobase-nucleobase interactions compared to unmodified tRNA or tRNA containing smaller (m1G) modifications at the 37th position. Importantly, the extent of the changes in the anticodon loop is influenced by the addition of small functional groups to parent modifications, implying each substituent can further fine-tune tRNA structure. Although the dominant conformation of the ASL is achieved in different ways for each modification, the molecular features of all modified tRNA drive the ASL domain to adopt the functional open-loop conformation. Importantly, the impact of the hypermodifications is preserved in different sequence contexts. These findings highlight the likely role of regulating mRNA structure and translation.

N6 -Isopentenyladenosine in RNA Determines the Cleavage Site of Endonuclease Deoxyribozymes.

RNA-cleaving deoxyribozymes can serve as selective sensors and catalysts to examine the modification state of RNA. However, site-specific endonuclease deoxyribozymes that selectively cleave post-transcriptionally modified RNA are extremely rare and their specificity over unmodified RNA is low. We report that the native tRNA modification N6 -isopentenyladenosine (i6 A) strongly enhances the specificity and has the power to reconfigure the active site of an RNA-cleaving deoxyribozyme. Using in vitro selection, we identified a DNA enzyme that cleaves i6 A-modified RNA at least 2500-fold faster than unmodified RNA. Another deoxyribozyme shows unique and unprecedented behaviour by shifting its cleavage site in the presence of the i6 A RNA modification. Together with deoxyribozymes that are strongly inhibited by i6 A, these results highlight that post-transcriptional RNA modifications modulate the catalytic activity of DNA in various intricate ways.

Chemical Deprenylation of N6 -Isopentenyladenosine (i6 A) RNA.

N6 -isopentenyladenosine (i6 A) is an RNA modification found in cytokinins, which regulate plant growth/differentiation, and a subset of tRNAs, where it improves the efficiency and accuracy of translation. The installation and removal of this modification is mediated by prenyltransferases and cytokinin oxidases, and a chemical approach to selective deprenylation of i6 A has not been developed. We show that a selected group of oxoammonium cations function as artificial deprenylases to promote highly selective deprenylation of i6 A in nucleosides, oligonucleotides, and live cells. Importantly, other epigenetic modifications, amino acid residues, and natural products were not affected. Moreover, a significant phenotype difference in the Arabidopsis thaliana shoot and root development was observed with incubation of the cation. These results establish these small organic molecules as direct chemical regulators/artificial deprenylases of i6 A.

Isolation and identification of N6-isopentenyladenosine as the cytotoxic constituent of a marine sponge Oceanapia sp.

N6-Isopentenyladenosine (i6A) was isolated from a marine sponge Oceanapia sp. as the major cytotoxic constituent along with N6-isopentenyladenosine 5'-monophosphate (i6AP) which was inactive. The structures of i6A and i6AP were assigned by a combination of the analysis of NMR spectroscopy and mass spectrometry. This is the first isolation of i6A and i6AP from a marine sponge.

The modified base isopentenyladenosine and its derivatives in tRNA.

Base 37 in tRNA, 3'-adjacent to the anticodon, is occupied by a purine base that is thought to stabilize codon recognition by stacking interactions on the first Watson-Crick base pair. If the first codon position forms an A.U or U.A base pair, the purine is likely further modified in all domains of life. One of the first base modifications found in tRNA is N6-isopentenyl adenosine (i6A) present in a fraction of tRNAs in bacteria and eukaryotes, which can be further modified to 2-methyl-thio-N6-isopentenyladenosine (ms2i6A) in a subset of tRNAs. Homologous tRNA isopentenyl transferase enzymes have been identified in bacteria (MiaA), yeast (Mod5, Tit1), roundworm (GRO-1), and mammals (TRIT1). In eukaryotes, isopentenylation of cytoplasmic and mitochondrial tRNAs is mediated by products of the same gene. Accordingly, a patient with homozygous mutations in TRIT1 has mitochondrial disease. The role of i6A in a subset of tRNAs in gene expression has been linked with translational fidelity, speed of translation, skewed gene expression, and non-sense suppression. This review will not cover the action of i6A as a cytokinin in plants or the potential function of Mod5 as a prion in yeast.

Methylated Cytokinins from the Phytopathogen Rhodococcus fascians Mimic Plant Hormone Activity.

Cytokinins (CKs), a class of phytohormones that regulate plant growth and development, are also synthesized by some phytopathogens to disrupt the hormonal balance and to facilitate niche establishment in their hosts. Rhodococcus fascians harbors the fasciation (fas) locus, an operon encoding several genes homologous to CK biosynthesis and metabolism. This pathogen causes unique leafy gall symptoms reminiscent of CK overproduction; however, bacterial CKs have not been clearly correlated with the severe symptoms, and no virulence-associated unique CKs or analogs have been identified. Here, we report the identification of monomethylated N(6)-(∆(2)-isopentenyl)adenine and dimethylated N(6)-(∆(2)-isopentenyl)adenine (collectively, methylated cytokinins [MeCKs]) from R. fascians. MeCKs were recognized by a CK receptor and up-regulated type-A ARABIDOPSIS THALIANA RESPONSE REGULATOR genes. Treatment with MeCKs inhibited root growth, a hallmark of CK action, whereas the receptor mutant was insensitive. MeCKs were retained longer in planta than canonical CKs and were poor substrates for a CK oxidase/dehydrogenase, suggesting enhanced biological stability. MeCKs were synthesized by S-adenosyl methionine-dependent methyltransferases (MT1 and MT2) that are present upstream of the fas genes. The best substrate for methylation was isopentenyl diphosphate. MT1 and MT2 catalyzed distinct methylation reactions; only the MT2 product was used by FAS4 to synthesize monomethylated N(6)-(∆(2)-isopentenyl)adenine. The MT1 product was dimethylated by MT2 and used as a substrate by FAS4 to produce dimethylated N(6)-(∆(2)-isopentenyl)adenine. Chemically synthesized MeCKs were comparable in activity. Our results strongly suggest that MeCKs function as CK mimics and play a role in this plant-pathogen interaction.

Biological Activity of Vegetal Extracts Containing Phenols on Plant Metabolism.

The influence of vegetal extracts derived from red grape, blueberry fruits and hawthorn leaves on Zea mays L. plant growth and the activity of phenylalanine ammonia-lyase (PAL), a key enzyme of the phenylpropanoid pathway, was investigated in laboratory experiments. The extracts were characterized using FT-IR and Raman spectroscopies in order to obtain a pattern of the main functional groups. In addition, phenols content was determined by HPLC, whereas the content of indoleacetic acid and isopentenyladenosine hormones was determined by ELISA test and the auxin and gibberellin-like activities by plant-bioassays. The treated maize revealed increased root and leaf biomass, chlorophyll and sugars content with respect to untreated plants. Hawthorn, red grape skin and blueberry at 1.0 mL/L induced high p-coumaric content values, whilst hawthorn also showed high amounts of gallic and p-hydroxybenzoic acids. PAL activity induced by hawthorn at 1.0 mL/L had the highest values (11.1-fold UNT) and was strongly and linearly related with the sum of leaf phenols. Our results suggest that these vegetal extracts contain more than one group of plant-promoting substances.

[Genome guided identification of 2-methylthio-N6-(4-hydroxy-isopentenyl)-adenosine from Streptomyces xinghaiensis NRRL B24674T].

Objective: To investigate the production of adenosine modified with N6-(Δ2-isopentenyl) and 2-thiomethyl groups from marine-derived Streptomyces xinghaiensis NRRL B24674T. Methods: Bioinformatics analysis was carried out to search the genome sequence of S. xinghaiensis NRRL B24674T and the secondary metabolites were purified by silica gel column chromatography, gel chromatography and high-performance liquid chromatography, and the chemical structure was elucidated by nuclear magnetic resonance (NMR) and mass spectroscopy (MS). Results: Two proteins involved in such a biosynthetic pathway were found in the genome of S. xinghaiensis NRRL B24674T; 2-methylthio-N6-(4-hydroxyisopentenyl)-adenosine has been purified from the liquid culture of S. xinghaiensis NRRL B24674T, and its chemical structure was elucidated by analysis of high-resolution mass spectrometry (HR-MS) and NMR data Conclusion: Such an adenine modification process was present in S. xinghaiensis NRRL B24674T, and it is the first time to report this kind of adenine modification from actinomycetes Streptomyces. Bioinformatics analysis implies that Streptomyces can also have this kind of RNA or adenine modification.

[Effects of local induction of ipt-gene in roots on cytokinins content in leaf cells tobacco plants].

Identification of cytokinins in differentiated leaf cells has received little attention. We have carried out immunohistochemical localization of cytokinins in leaves of transgenic tobacco plants in which the level of increased due to induced in their roots the expression of ipt-gene controlling cytokinin synthesis. Immuno-labeling of cytokinins with the help of antibodies raised against zeatin riboside was characteristic of mesophyll cells. The label was localized in cytoplasm adjacent to cell walls and was absent in vacuoles. Immunohistochemical staining also revealed the presence of cytokinins in guard cells. Induction of cytokinin synthesis enhanced the immunohistochemical staining of both mesophyll cells and guard cells, which was accompanied by elevated stomatal conductance. The possibility of a direct effect of cytokinins on stomatal conductance and their indirect influence through photosynthesis in the mesophyll cells is discussed.

Sensitive quantification of isoprenoid cytokinins in plants by selective immunoaffinity purification and high performance liquid chromatography-quadrupole-time of flight mass spectrometry.

In this work, we developed a novel class-specific immunoaffinity column for the natural isoprenoid cytokinins (CTKs) by using trans-zeatin riboside as the hapten to generate a complete antigen. By combination with a mixed-mode solid phase extraction step for pre-cleanup and a high performance liquid chromatography-quadrupole-time of flight mass spectrometry for the quantification, an efficient analytical protocol was established which allowed simultaneous quantification of eight endogenous isoprenoid CTKs in Arabidopsis thaliana leaves with a wide linear range from 25 to 500pg/g fresh weight and a detection limit of 12.5pg/g fresh weight. The method will be very useful for comprehensive research on the networks of signaling interactions of the active phytohormones and their regulation of the plant functions.

Transformations of the natural cytokinin N6-isopentenyladenine in aqueous acidic media: structural aspects.

N6-Isopentenyladenine (L1) was subjected to variously acidic media in 0.1 M, 1 M and 2 M HCl. In dependence on the acidity of the medium, the formation of three main acid hydrolysis products, involving the N6-isopentenyladeninium (HL1) (1), 7,8,9,10-tetrahydro-7,7-dimethyl-3H-pyrimido[2,1-i]purin-6-ium (HL2) (2) or 5-amino-4-(4,4-dimethyl-3,4,5,6-tetrahydropyrimidin-2-yl)-imidazolium (H(2)L3) (3-5) cations, were determined and characterized by multinuclear solution-state NMR spectroscopy and in the solid state by single crystal X-ray analysis. The coordination abilities of these transformation products have been also investigated. The compounds of the compositions [Zn(HL1)Cl(3)]·H(2)O (1), [Zn(3)(HL2)(2)Cl(8)] (2), (H(2)L3)[CuCl(4)] (4) and (H(2)L3)[ZnCl(4)] (5) have been prepared in dependence on the acidity of the medium used by the reactions of L1 with ZnCl(2)·1.5H(2)O or CuCl(2)·2H(2)O. Based on the NMR spectroscopic and X-ray crystallographic results, the mechanism of transformation of L1 in the acidic medium, involving the protonation, cyclization and ring fission, has been suggested.

Synthesis and evaluation of in vitro anticancer activity of some novel isopentenyladenosine derivatives.

The present study describes the synthesis, the characterization and the evaluation of some derivatives of N(6)-isopentenyladenosine on T24 human bladder carcinoma cells. In particular we have modified the hydroxyl groups in the ribose moiety, the position of the isopentenyl chain in the purine ring and the base moiety. The structures of the compounds were confirmed by standard studies of NMR, MS and elemental analysis. We here show that only two derivatives, 1-(3-methyl-2-butenylamino)-9-(3'-deoxy-beta-d-ribofuranosyl)-purine hydrobromide and 2-amino-6-(3-methyl-2- butenylamino)-9-(beta-d-ribofuranosyl)-purine, inhibit the growth of T24 cells, although to a lower extent than N(6)-isopentenyladenosine. We conclude that the integrity of ribosidic and purine moiety and the N(6) position of the chain are essential for maintaining the antiproliferative activity.

1H, 13C and 15N NMR assignments for N6-isopentenyladenosine/inosine analogues.

The complete (1)H, (13)C and (15)N NMR signals assignments of some new isopentenyladenosine analogues were achieved using one- and two-dimensional experiments (gs-NOESY, gs-HMQC and gs-HMBC).

Pharmacogenomics and analogues of the antitumour agent N6-isopentenyladenosine.

N(6)-isopentenyladenosine (i(6)A), a member of the cytokinin family of plant hormones, has potent in vitro antitumour activity in different types of human epithelial cancer cell lines. Gene expression profile analysis of i(6)A-treated cells revealed induction of genes (e.g., PPP1R15A, DNAJB9, DDIT3, and HBP1) involved in the negative regulation of cell cycle progression and reportedly up-regulated during cell cycle arrest in stress conditions. Of 6 i(6)A analogues synthesized, only the 1 with a saturated double bond of the isopentenyl side chain had in vitro antitumour activity, although weaker than that of i(6)A, suggesting that i(6)A biological activity is highly linked to its structure. In vivo analysis of i(6)A and the active analogue revealed no significant inhibition of cancer cell growth in mice by either reagent. Thus, although i(6)A may inhibit cell proliferation by regulating the cell cycle, further studies are needed to identify active analogues potentially useful in vivo.

Design, synthesis and perception of fluorescently labeled isoprenoid cytokinins.

Isoprenoid cytokinins play a number of crucial roles in the regulation of plant growth and development. To study cytokinin receptor properties in plants, we designed and prepared fluorescent derivatives of 6-[(3-methylbut-2-en-1-yl)amino]purine (N6-isopentenyladenine, iP) with several fluorescent labels attached to the C2 or N9 atom of the purine moiety via a 2- or 6-carbon linker. The fluorescent labels included dansyl (DS), fluorescein (FC), 7-nitrobenzofurazan (NBD), rhodamine B (RhoB), coumarin (Cou), 7-(diethylamino)coumarin (DEAC) and cyanine 5 dye (Cy5). All prepared compounds were screened for affinity for the Arabidopsis thaliana cytokinin receptor (CRE1/AHK4). Although the attachment of the fluorescent labels to iP via the linkers mostly disrupted binding to the receptor, several fluorescent derivatives interacted well. For this reason, three derivatives, two rhodamine B and one 4-chloro-7-nitrobenzofurazan labeled iP were tested for their interaction with CRE1/AHK4 and Zea mays cytokinin receptors in detail. We further showed that the three derivatives were able to activate transcription of cytokinin response regulator ARR5 in Arabidopsis seedlings. The activity of fluorescently labeled cytokinins was compared with corresponding 6-dimethylaminopurine fluorescently labeled negative controls. Selected rhodamine B C2-labeled compounds 17, 18 and 4-chloro-7-nitrobenzofurazan N9-labeled compound 28 and their respective negative controls (19, 20 and 29, respectively) were used for in planta staining experiments in Arabidopsis thaliana cell suspension culture using live cell confocal microscopy.

N6-isopentenyladenosine dual targeting of AMPK and Rab7 prenylation inhibits melanoma growth through the impairment of autophagic flux.

Targeting the autophagic process is considered a promising therapeutic strategy in cancer since a great number of tumors, including melanoma, show high basal levels of protective autophagy that contributes to tumor progression and chemoresistance. Here, exploiting both in vitro and in vivo approaches, we identified N6-isopentenyladenosine (iPA), an end product of the mevalonate pathway, as a novel autophagy inhibitor with an interesting anti-melanoma activity. iPA, after being phosphorylated by adenosine kinase into 5'-iPA-monophosphate, induces autophagosome accumulation through AMPK activation, measured by increased fluorescent GFP-LC3 puncta and enhanced conversion into the lipidated autophagosome-associated LC3-II. However, at a later stage iPA blocks the autophagic flux monitored by p62 accumulation, Luciferase reporter-based assay for LC3 turnover in living cells and fluorescence of a tandem RFP-GFP-LC3 construct. Impaired autophagic flux is due to the block of autophagosome-lysosome fusion through the defective localization and function of Rab7, whose prenylation is inhibited by iPA, resulting in a net inhibition of autophagy completion that finally leads to melanoma apoptotic cell death. AMPK silencing prevents apoptosis upon iPA treatment, whereas basal autophagosome turnover is still inhibited due to unprenylated Rab7. These results strongly support the advantage of targeting autophagy for therapeutic gain in melanoma and provide the preclinical rational to further investigate the antitumor action of iPA, able to coordinately induce autophagosome accumulation and inhibit the autophagic flux, independently targeting AMPK and Rab7 prenylation. This property may be particularly useful for the selective killing of tumors, like melanoma, that frequently develop chemotherapy resistance due to protective autophagy activation.

tRNA anticodons with the modified nucleoside 2-methylthio-N6-(4-hydroxyisopentenyl)adenosine distinguish between bases 3' of the codon.

The modified nucleoside 2-methylthio-N6-(4-hydroxyisopentenyl)adenosine (ms2io6A) is present immediately to the 3' side of the anticodon (position 37) in tRNAs that read codons starting with uridine and hence include amber (UAG) suppressor tRNAs. We have used strains of Salmonella typhimurium that differ only in their ability to synthesize ms2io6A in order to determine specifically how this modified nucleoside influences the efficiency of amber suppression in two codon contexts differing by only which base is 3' of the codon. The results show that the presence of the modified nucleoside ms2io6A not only improves the efficiency of the suppressor tRNAs but also allows them to distinguish between at least two bases 3' of the codon. Thus, the presence of ms2io6A reduces the intrinsic codon context sensitivity of the tRNA and specifically counteracts an unfavourable nucleotide on the 3' side of the codon. The possible codon-anticodon interactions responsible for this effect are discussed.