Methylated Nucleosides Research Articles

Robust Bisulfite‐Free Single‐Molecule Real‐Time Sequencing of Methyldeoxycytidine Based on a Novel hpTet3 Enzyme

In addition to the four canonical nucleosides dA, dG, dC and T, genomic DNA contains the additional base 5‐methyldeoxycytidine (mdC). The presence of this methylated cytidine nucleoside in promoter regions or gene bodies significantly affects the transcriptional activity of the corresponding gene. Consequently, the methylation patterns of genes are crucial for either silencing or activating genes. Sequencing the positions of mdC in the genome is therefore of paramount importance for early cancer diagnostics as it helps determine incorrect gene expression. Currently, the bisulfite method is the gold standard for mdC‐sequencing. However, this method has the drawback that the majority of the input DNA is degraded during the bisulfite treatment. Additionally, bisulfite sequencing is prone to errors. Here, we report a benign, bisulfite‐free mdC sequencing method termed EMox‐seq, which is based on third‐generation single‐molecule SMRT sequencing. The foundation of this technology is a new Tet3 enzyme that efficiently oxidizes mdCs to 5‐carboxycytidine (cadC). In turn, cadC provides an excellent readout by SMRT sequencing using specially trained AI‐based algorithms.

Robust Bisulfite-Free Single-Molecule Real-Time Sequencing of Methyldeoxycytidine Based on a Novel hpTet3 Enzyme.

In addition to the four canonical nucleosides dA, dG, dC and T, genomic DNA contains the additional base 5-methyldeoxycytidine (mdC). The presence of this methylated cytidine nucleoside in promoter regions or gene bodies significantly affects the transcriptional activity of the corresponding gene. Consequently, the methylation patterns of genes are crucial for either silencing or activating genes. Sequencing the positions of mdC in the genome is therefore of paramount importance for early cancer diagnostics as it helps determine incorrect gene expression. Currently, the bisulfite method is the gold standard for mdC-sequencing. However, this method has the drawback that the majority of the input DNA is degraded during the bisulfite treatment. Additionally, bisulfite sequencing is prone to errors. Here, we report a benign, bisulfite-free mdC sequencing method termed EMox-seq, which is based on third-generation single-molecule SMRT sequencing. The foundation of this technology is a new Tet3 enzyme that efficiently oxidizes mdCs to 5-carboxycytidine (cadC). In turn, cadC provides an excellent readout by SMRT sequencing using specially trained AI-based algorithms.

Accurate Quantification of Ten Methylated Purine Nucleosides by Highly Sensitive and Stable Isotope-Diluted UHPLC-MS/MS.

The dynamic landscape of cellular nucleotides/nucleosides associated with RNA metabolism, particularly in diseases like cancer, has spurred intensive interest. Here, we report a robust stable isotope-diluted UHPLC-ESI-MS/MS method for accurate quantification of 12 purine ribonucleosides, including 10 methylated purine nucleosides. By the use of thermally decomposable ammonium bicarbonate (NH4HCO3) as a mobile phase additive for UHPLC-MS/MS detection, the ESI-MS/MS signal responses of these target compounds were enhanced by 1.7-24.5 folds. Noteworthily, three methylated guanosine isomers (m1G, m2G, and m7G) and two methylated adenosine isomers (m1A and m6A) that are indistinguishable directly by mass spectrometry were well resolved with optimal UHPLC separation. Combined with methanol extraction and solid-phase extraction (SPE) pretreatment, the method quantified intracellular concentrations of three modified nucleosides (Gm, m1G, and m2G), which would otherwise be undetectable because of significant suppression of their signals by the interfering cellular matrix. Nine purine nucleosides were simultaneously quantified in 293T cells, and their concentrations ranged by 4 orders of magnitude. Overall, the method presents high recovery rates over 90% for endogenous modified purine nucleosides in cultured cells, along with good precision, linearity, and LOD ranging from 0.30 fmol to 0.37 pmol per 5 × 105 cells. The developed UHPLC-MS/MS method holds potential for screening purine nucleosides as diagnostic and prognostic biomarkers and for quantifying purine epigenetic nucleosides post-cell metabolome analysis, thereby providing a valuable analytical tool for intracellular nucleoside quantification in future clinical research.

Aminodealkenylation: Ozonolysis and copper catalysis convert C(sp3)-C(sp2) bonds to C(sp3)-N bonds.

Great efforts have been directed toward alkene π bond amination. In contrast, analogous functionalization of the adjacent C(sp3)-C(sp2) σ bonds is much rarer. Here we report how ozonolysis and copper catalysis under mild reaction conditions enable alkene C(sp3)-C(sp2) σ bond-rupturing cross-coupling reactions for the construction of new C(sp3)-N bonds. We have used this unconventional transformation for late-stage modification of hormones, pharmaceutical reagents, peptides, and nucleosides. Furthermore, we have coupled abundantly available terpenes and terpenoids with nitrogen nucleophiles to access artificial terpenoid alkaloids and complex chiral amines. In addition, we applied a commodity chemical, α-methylstyrene, as a methylation reagent to prepare methylated nucleosides directly from canonical nucleosides in one synthetic step. Our mechanistic investigation implicates an unusual copper ion pair cooperative process.

The application of TMAH thermochemolysis on the detection of nucleotides: applications for the SAM and MOMA space experiments

The Sample Analysis at Mars (SAM) instrument onboard the Curiosity rover and Mars Organic Molecule Analyzer (MOMA) instrument onboard the ExoMars rover are two of the most important instruments used for the in-situ search for biosignatures of life on Mars. Tetramethylammonium hydroxide (TMAH) thermochemolysis combined with pyrolysis-gas chromatography and mass spectrometry (Py-GC/MS) has been one of the main techniques utilized by SAM and will be utilized by MOMA. They are both capable of detecting molecular fragments and patterns indicative of life in the Martian near-surface. This study identifies the TMAH thermochemolysis products of targeted nucleotides using flash pyrolysis and SAM-like ramp pyrolysis experiments. Additionally, the optimal TMAH thermochemolysis temperature was determined. Results indicate that the methylated nucleosides can be detected at low abundances when exposed to TMAH thermochemolysis flash pyrolysis at 200 °C. Notably, higher pyrolysis temperatures led to the degradation of nucleosides. Furfuryl methyl ether, one of the degradation products of these nucleosides, was also identified from 200 to 600°C in this study. 300 °C is the optimal temperature for the detection of methylated phosphate under flash pyrolysis of the tested nucleotides with TMAH thermochemolysis. Methylated nucleobases, methyl furfuryl and methyl phosphate are the main products of the tested nucleotides under SAM-like ramp thermochemolysis. Ramped thermochemolysis also resulted in improved performance over flash pyrolysis in the detection of characteristic nucleotide compounds. Uracil and thymine can be detected by both SAM and MOMA if there are nucleotides on Mars; additionally, MOMA will be able to detect adenine. Therefore, methyl furfuryl, methylated phosphate, and the methylated nucleobases uracil, thymine, and adenine are the organic compounds that should be within the SAM and MOMA detection windows if there is DNA/RNA conserved in Martian soil. These experiments will provide a reference for the data collected in-situ by the Curiosity rover and the future ExoMars rover.

The application of TMAH thermochemolysis on the detection of nucleosides: Applications for the SAM and MOMA instruments

Tetramethylammonium hydroxide (TMAH) thermochemolysis has been utilized by the Curiosity rover’s Sample Analysis at Mars (SAM) instrument and will be utilized by the ExoMars 2022′s Mars Organic Analyzer (MOMA) instrument. TMAH thermochemolysis is one of the main techniques that enables the detection of bioindicators when it is combined with pyrolysis-gas chromatography and mass spectrometry (Py-GC/MS) at the surface of Mars. This study identifies the thermochemolysis products of targeted nucleosides using flash pyrolysis and a SAM-like ramp pyrolysis. Results indicate that the methylated nucleosides can be detected when nucleosides are submitted to TMAH thermochemolysis, which demonstrates that the TMAH thermochemolysis protocol does not result in the decomposition of the nucleosides. To detect the whole structure of each nucleoside, low thermochemolysis temperature is required. 200 °C is the optimal temperature. Methylated nucleobases are the main products of the tested nucleosides when the thermochemolysis temperature is higher than 300 °C. Furfuryl methyl ether, one of the degradation products of these nucleosides, were also identified from 200° to 600°C in this study. These experiments will establish a reference database for interpretation of the data acquired by the SAM and MOMA experiments during operations on Mars.

Simultaneous Determination of Methylated Nucleosides by HILIC-MS/MS Revealed Their Alterations in Urine from Breast Cancer Patients.

RNA methylation plays a vital role in the pathogenesis of a variety of diseases including cancer, and aberrant levels of modified nucleosides in RNA were revealed to be related to cancer. Urine is a favored source for biomarker discovery due to the non-invasion to patients. Herein, we developed a sensitive hydrophilic interaction liquid chromatography tandem mass spectrometry (HILIC–MS/MS) method combined with stable isotope dilution for accurate quantification of methylated nucleosides in human urine. With this method, we successfully quantified ten methylated nucleosides in urine samples collected from healthy controls and breast cancer patients. We found N6-methyladenosine (m6A), 2′-O-methyladenosine (Am), N1-methyladenosine (m1A), N6,2′-O-dimethyladenosine (m6Am), N1-methylguanosine (m1G), 2′-O-methylguanosine (Gm), 5-methylcytidine (m5C) and 2′-O-methylcytidine (Cm) were all decreased in early-stage breast cancer patients, and a nomogram prediction model was constructed. Locally advanced breast cancer patients exhibited elevated levels of urinary 2′-O-methylated nucleosides in comparison to early-stage breast cancer patients. Together, we developed a robust method for the simultaneous determination of methylated nucleosides in human urine, and the results revealed an association between the contents of urinary methylated nucleosides and the occurrence of breast cancer, which may stimulate future studies about the regulatory roles of these methylated nucleosides in the initiation and progression of breast cancer.

Determination of adenosine and its modifications in urine and plasma from breast cancer patients by hydrophilic interaction liquid chromatography-tandem mass spectrometry

RNA modifications have been revealed to be essential in many biological activities, and their disorders are associated with various human diseases, including cancers. 2′-O-methyladenosine (Am), N1-methyladenosine (m1A), N6-methyladenosine (m6A), N6,2′-O-dimethyladenosine (m6Am) and N6,N6-dimethyladenosine (m62A) are important adenosine (A) modifications. The noninvasive collection of urine samples and the diverse contents of metabolites in plasma make them favored biofluids for biomarkers discovery. In this work, we established a hydrophilic interaction liquid chromatography-tandem mass spectrometry (HILIC-MS/MS) method to quantify these six nucleosides in urine and plasma of healthy controls and breast cancer (BC) patients. The limit of detection (LOD) for A, Am, m1A, m6A, m6Am, and m62A were 0.0025, 0.01, 0.05, 0.005, 0.005, and 0.005 nM. The results showed that the concentrations of Am, m6A, and m6Am were increased, whereas m1A was decreased in the urine of BC patients compared with the healthy controls. We also found that the level ratios of m1A/A, m6A/A, and m6Am/A were all reduced in plasma from BC patients, compared with healthy controls. Interestingly, these ratios of methylated adenosine nucleosides to adenosine in plasma could better discriminate BC patients from healthy controls, compared to the levels of these nucleosides. The present study not only suggests these modified adenosines can act as noninvasive biomarkers of BC but also will contribute to investigating the impacts of RNA methylation on the occurrence and development of BC.

RBM45 is an m6A-binding protein that affects neuronal differentiation and the splicing of a subset of mRNAs.

SUMMARYN6-methyladenosine (m6A) is deposited co-transcriptionally on thousands of cellular mRNAs and plays important roles in mRNA processing and cellular function. m6A is particularly abundant within the brain and is critical for neurodevelopment. However, the mechanisms through which m6A contributes to brain development are incompletely understood. RBM45 acts as an m6A-binding protein that is highly expressed during neurodevelopment. We find that RBM45 binds to thousands of cellular RNAs, predominantly within intronic regions. Rbm45 depletion disrupts the constitutive splicing of a subset of target pre-mRNAs, leading to altered mRNA and protein levels through both m6A-dependent and m6A-independent mechanisms. Finally, we find that RBM45 is necessary for neuroblastoma cell differentiation and that its depletion impacts the expression of genes involved in several neurodevelopmental signaling pathways. Altogether, our findings show a role for RBM45 in controlling mRNA processing and neuronal differentiation, mediated in part by the recognition of methylated RNA.

Honeybee Iflaviruses Pack Specific tRNA Fragments from Host Cells in Their Virions.

The Picornavirales include viruses that infect vertebrates, insects, and plants. It was believed that they pack only their genomic mRNA in the particles; thus, we envisaged these viruses as excellent model systems for studies of mRNA modifications. We used LC–MS to analyze digested RNA isolated from particles of the sacbrood and deformed wing iflaviruses as well as of the echovirus 18 and rhinovirus 2 picornaviruses. Whereas in the picornavirus RNAs we detected only N 6‐methyladenosine and 2’‐O‐methylated nucleosides, the iflavirus RNAs contained a wide range of methylated nucleosides, such as 1‐methyladenosine (m1A) and 5‐methylcytidine (m5C). Mapping of m1A and m5C through RNA sequencing of the SBV and DWV RNAs revealed the presence of tRNA molecules. Both modifications were detected only in tRNA. Further analysis revealed that tRNAs are present in form of 3’ and 5’ fragments and they are packed selectively. Moreover, these tRNAs are typically packed by other viruses.

Physicochemical properties calculated using DFT method and changes of 5-methyluridine hemihydrate crystals at high temperatures

5-methyluridine hemihydrate (5 mU) single crystals were synthesized by the slow solvent evaporation method. The physicochemical properties, such as frontier molecular orbitals, global reactivity indices and vibrational were computationally studied through density functional theory (DFT). In addition, structural, vibrational, and thermal properties were obtained by powder X-ray diffraction (PXRD), Raman spectroscopy, thermogravimetric (TG) analysis and differential scanning calorimetry (DSC). PXRD evaluated the structural behavior of 5 mU crystal in the temperature range of 300–460 K. The high-temperature PXRD results suggested that the crystal undergoes two dehydration processes, being a first occurring from the orthorhombic structure (P21212) to triclinic (P1), in which the water losses occurred around 380 K. A second dehydration triggers the change from the triclinic structure to monoclinic (P21) within the 420–435 K temperature range. Furthermore, after this temperature, the anhydrous 5 mU suffers a melting process near 460 K, which is remarkably characterized as an irreversible process. Raman spectroscopy was carried out to identify the vibrational modes linked to the water molecule and the noticeable changes in these bands due to high-temperature effects around 380 K and 410 K. Indeed, changes on Raman bands, such as intensity inversion, the disappearance of bands associated with the hydrogen bonds formed from the water molecules and uracil group, and the ribose group were observed. Finally, this study provided details on the structural and vibrational changes caused by the dehydration of 5 mU crystals and the importance of hydrogen bonds for understanding the intermolecular interactions of the 5 mU, a methylated nucleoside with important biological functions.

HILIC-MS/MS for the Determination of Methylated Adenine Nucleosides in Human Urine.

N6-methyl-2'-deoxyadenosine (m6dA) is a newly discovered DNA epigenetic mark in mammals. N6-methyladenosine (m6A), 2'-O-methyladenosine (Am), N6,2'-O-dimethyladenosine (m6Am), and N6,N6-dimethyladenosine (m62A) are common RNA modifications. Previous studies illustrated the associations between the aberrations of these methylated adenosines in nucleic acids and cancer. Herein, we developed Fe3O4/graphene-based magnetic dispersive solid-phase extraction for the enrichment and hydrophilic interaction liquid chromatography-mass spectrometry (HILIC-MS/MS) for the measurements of m6dA, m6A, Am, m6Am, and m62A in human urine samples. We found that malic acid could improve the HILIC-based separation of these modified nucleosides and markedly enhance the sensitivity of their MS detection. With this method, we accurately quantified the contents of these modified adenine nucleosides in urine samples collected from gastric and colorectal cancer patients as well as healthy controls. We found that, relative to healthy controls, urinary m6dA and Am levels are significantly lower for gastric and colorectal cancer patients; while gastric cancer patients also exhibited lower levels of urinary m6A, the trend was opposite for colorectal cancer patients. Together, we developed a robust analytical method for simultaneous measurements of five methylated adenine nucleosides in human urine, and our results revealed an association between the levels of urinary methylated adenine nucleosides and the occurrence of gastric as well as colorectal cancers, suggesting the potential applications of these modified nucleosides as biomarkers for the early detection of these cancers.

CPA-seq reveals small ncRNAs with methylated nucleosides and diverse termini

High-throughput sequencing reveals the complex landscape of small noncoding RNAs (sRNAs). However, it is limited by requiring 5′-monophosphate and 3′-hydroxyl in RNAs for adapter ligation and hindered by methylated nucleosides that interfere with reverse transcription. Here we develop Cap-Clip acid pyrophosphatase (Cap-Clip), T4 polynucleotide kinase (PNK) and AlkB/AlkB(D135S)-facilitated small ncRNA sequencing (CPA-seq) to detect and quantify sRNAs with terminus multiplicities and nucleoside methylations. CPA-seq identified a large number of previously undetected sRNAs. Comparison of sRNAs with or without AlkB/AlkB(D135S) treatment reveals nucleoside methylations on sRNAs. Using CPA-seq, we profiled the sRNA transcriptomes (sRNomes) of nine mouse tissues and reported the extensive tissue-specific differences of sRNAs. We also observed the transition of sRNomes during hepatic reprogramming. Knockdown of mesenchymal stem cell-enriched U1-5′ snsRNA promoted hepatic reprogramming. CPA-seq is a powerful tool with high sensitivity and specificity for profiling sRNAs with methylated nucleosides and diverse termini.

Discrimination of common isomerides of methyl nucleosides by collision-induced dissociation tandem mass spectrometry.

The methyl-modified nucleosides (methyladenosine, methylguanosine, methylcytidine, and methyluridine) occur widely in nucleic acids and serve as biomarkers for a variety of types of diseases. Their isomers have the same parent ions in MS spectra and need further discrimination. Here, electrospray ionization mass spectrometry (ESI-MS) coupled with collision induced dissociation (CID) was used to distinguish the common isomerides of methyl-nucleosides. The various fragmentation patterns had been discussed in comparison of the different methyl modified nucleosides and were studied as a function of normalized collision energy. Then, structurally relevant fragments were obtained to efficiently identify characterization of methyl-nucleoside isomerides by collision induced dissociation. Therefore, this study provides a promising method using CID-MS for the discrimination of the isomeric methyl nucleosides, which could be useful to quantitative study of methyl nucleosides and detect of unknown methyl nucleosides.

Expanding Mass Spectrometry Toolbox for RNA Modification Profiling

RNA modifications, by expanding the repertoire of RNA structure and interactions, play important roles in biology. At the cellular level, modifications have been linked to antibiotic resistance, RNA biogenesis, translational fidelity, and gene expression. Importantly, stoichiometry of many RNA modifications is responsive to cellular and environmental cues, and can vary across cell types and disease states. Here we present the experimental and bioinformatic tools for mass spectrometry (MS) based identification and quantitative profiling of RNA modifications in abundant RNAs. RNA modification analysis is based on nucleolytic cleavage of the purified RNA of interest into small 3-15 nt fragments that are chromatographically separated and sequenced via LC-MS/MS. Compared to the field of protein identification, analysis of the tandem MS spectra of RNA has been significantly lagging behind, due to more complex fragmentation patterns and high redundancy of RNA (4 nt vs. 20 aa in proteomics). To address the need we developed Pytheas, a bioinformatic software for annotation of RNA MS/MS spectra and assignment to the sequences present in the theoretical library. Pytheas is using an empirical scoring function that has been trained to successfully distinguish between the target and decoy sequences and is equipped with statistical tools for the automated analysis at the large scale. Pytheas performance has been tested using MS/MS data collected on different instruments and RNAs of different origin, including in-vitro transcribed RNA, rRNA, and tRNA. As an example, we will demonstrate the identification coverage of known ribosomal modifications in S. Cerevisiae. Additionally, 16S and 23S isolated from B. Subtilis will be used to show how Pytheas can be implemented for the discovery and sequence placement of previously unannotated methylated nucleosides and “mass-silent” pseudouridines using both stable-isotope labeling with 2H and 15N, and chemical-derivatization.

Development and validation of a rapid LC–MS/MS method for determination of methylated nucleosides and nucleobases in urine

Modified nucleosides and nucleobases serve as potential diagnostic and prognostic markers of various diseases, including cancer. These compounds are hydrophilic, but are determined mainly using columns with C18 stationary phase. Moreover, these compounds require purification due to the presence of high amounts of isotopomers in the sample matrix and risk of matrix interferences. The most commonly used method for analyte extraction (i.e. solid-phase extraction on phenylboronic acid sorbent) is not appropriate for all the analytes since compounds with cis-diol groups (e.g. 7-methylguanine) are not absorbed. Thus, the aim of this study was to develop and validate a simple and fast method for the simultaneous determination of the methylated nucleosides and nucleobases (derivatives of adenine and guanine including those with cis-diol groups) in urine. The method was based on hydrophilic interaction liquid chromatography coupled with tandem mass spectrometry (HILIC–MS/MS). The sample preparation involved only dilution with acetonitrile and centrifugation. The method was validated for selectivity, calibration curve, precision, accuracy, stability, matrix effect, dilution integrity, and carryover, according to the guidelines of the European Medicines Agency (EMA). All the analyzed validation criteria were fulfilled. The method was applied to the urine of rats and humans (adults and children). To our best knowledge, HILIC-MS/MS method established by our team is the first method that does not require the extraction step as well as this method enables simultaneously determination of 1-methylguanine, 2′-O-methylguanosine, 3-methyladenine, and N6-methyl-2′-deoxyadenosine in urine. The method is reliable and can be applied to determine the concentrations of the modified nucleosides and nucleobases in the urine of humans and rats. Because the method is cost-effective, fast, and easy to perform, it may be considered as a routine tool in clinical laboratories.

Determination of RNA Hydroxylmethylation in Mammals by Mass Spectrometry Analysis

RNA molecules harbor diverse chemical modifications that play important regulatory roles in a variety of biological processes. Up to date, more than 150 modifications have been identified in various RNA species. Most of these modifications occurring in nucleic acids are the methylation of nucleic acids. It has been demonstrated that many of these methylation are reversible and undergo dynamic demethylation. Previous studies established that the demethylation of the two most important and prevalent modifications of 5-methylcytidine (m5C) and N6-methyladenosine (m6A) in nucleic acids is through the hydroxylation of m5C and m6A, forming 5-hydroxymethylcytidine (hm5C) and N6-hydroxymethyladenosine (hm6A), respectively. This indicates the hydroxylation of the methylated nucleosides may be a general pathway for the demethylation of nucleic acid methylation. However, few other hydroxylmethylation modifications have yet to be reported in existence in mammals. In the current study, we developed a neutral enzymatic digestion method for the mild digestion of nucleic acids, followed by liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS) analysis. With the established method, we reported the existence of a new hydroxylmethylated nucleosides, N2-hydroxymethylguanosine (hm2G), in mammalian RNA. In addition, we found that the contents of hm2G, as well as N2-methylguanosine (m2G), showed significant differences between thyroid carcinoma tissues and tumor-adjacent normal tissues, indicating that m2G and hm2G in RNA may play certain roles in the carcinogenesis of thyroid carcinoma. Collectively, our study suggests that RNA hydroxylmethylation may be a new prevalent group of modifications existing in RNA, which expands the diversity of nucleic acid modifications and should exert regulatory functions in living organisms.

Sources of artifact in measurements of 6mA and 4mC abundance in eukaryotic genomic DNA

BackgroundDirected DNA methylation on N6-adenine (6mA), N4-cytosine (4mC), and C5-cytosine (5mC) can potentially increase DNA coding capacity and regulate a variety of biological functions. These modifications are relatively abundant in bacteria, occurring in about a percent of all bases of most bacteria. Until recently, 5mC and its oxidized derivatives were thought to be the only directed DNA methylation events in metazoa. New and more sensitive detection techniques (ultra-high performance liquid chromatography coupled with mass spectrometry (UHPLC-ms/ms) and single molecule real-time sequencing (SMRTseq)) have suggested that 6mA and 4mC modifications could be present in a variety of metazoa.ResultsHere, we find that both of these techniques are prone to inaccuracies, which overestimate DNA methylation concentrations in metazoan genomic DNA. Artifacts can arise from methylated bacterial DNA contamination of enzyme preparations used to digest DNA and contaminating bacterial DNA in eukaryotic DNA preparations. Moreover, DNA sonication introduces a novel modified base from 5mC that has a retention time near 4mC that can be confused with 4mC. Our analyses also suggest that SMRTseq systematically overestimates 4mC in prokaryotic and eukaryotic DNA and 6mA in DNA samples in which it is rare. Using UHPLC-ms/ms designed to minimize and subtract artifacts, we find low to undetectable levels of 4mC and 6mA in genomes of representative worms, insects, amphibians, birds, rodents and primates under normal growth conditions. We also find that mammalian cells incorporate exogenous methylated nucleosides into their genome, suggesting that a portion of 6mA modifications could derive from incorporation of nucleosides from bacteria in food or microbiota. However, gDNA samples from gnotobiotic mouse tissues found rare (0.9–3.7 ppm) 6mA modifications above background.ConclusionsAltogether these data demonstrate that 6mA and 4mC are rarer in metazoa than previously reported, and highlight the importance of careful sample preparation and measurement, and need for more accurate sequencing techniques.

Identification of a 2′-O-Methyluridine Nucleoside Hydrolase Using the Metagenomic Libraries

Ribose methylation is among the most ubiquitous modifications found in RNA. 2′-O-methyluridine is found in rRNA, snRNA, snoRNA and tRNA of Archaea, Bacteria, and Eukaryota. Moreover, 2′-O-methylribonucleosides are promising starting materials for the production of nucleic acid-based drugs. Despite the countless possibilities of practical use for the metabolic enzymes associated with methylated nucleosides, there are very few reports regarding the metabolic fate and enzymes involved in the metabolism of 2′-O-alkyl nucleosides. The presented work focuses on the cellular degradation of 2′-O-methyluridine. A novel enzyme was found using a screening strategy that employs Escherichia coli uracil auxotroph and the metagenomic libraries. A 2′-O-methyluridine hydrolase (RK9NH) has been identified together with an aldolase (RK9DPA)—forming a part of a probable gene cluster that is involved in the degradation of 2′-O-methylated nucleosides. The RK9NH is functional in E. coli uracil auxotroph and in vitro. The RK9NH nucleoside hydrolase could be engineered to enzymatically produce 2′-O-methylated nucleosides that are of great demand as raw materials for production of nucleic acid-based drugs. Moreover, RK9NH nucleoside hydrolase converts 5-fluorouridine, 5-fluoro-2′-deoxyuridine and 5-fluoro-2′-O-methyluridine into 5-fluorouracil, which suggests it could be employed in cancer therapy.

Identification of tRNA nucleoside modification genes critical for stress response and development in rice and Arabidopsis

BackgroundModification of nucleosides on transfer RNA (tRNA) is important either for correct mRNA decoding process or for tRNA structural stabilization. Nucleoside methylations catalyzed by MTase (methyltransferase) are the most common type among all tRNA nucleoside modifications. Although tRNA modified nucleosides and modification enzymes have been extensively studied in prokaryotic systems, similar research remains preliminary in higher plants, especially in crop species, such as rice (Oryza sativa). Rice is a monocot model plant as well as an important cereal crop, and stress tolerance and yield are of great importance for rice breeding.ResultsIn this study, we investigated how the composition and abundance of tRNA modified nucleosides could change in response to drought, salt and cold stress, as well as in different tissues during the whole growth season in two model plants–O. sativa and Arabidopsis thaliana. Twenty two and 20 MTase candidate genes were identified in rice and Arabidopsis, respectively, by protein sequence homology and conserved domain analysis. Four methylated nucleosides, Am, Cm, m1A and m7G, were found to be very important in stress response both in rice and Arabidopsis. Additionally, three nucleosides,Gm, m5U and m5C, were involved in plant development. Hierarchical clustering analysis revealed consistency on Am, Cm, m1A and m7G MTase candidate genes, and the abundance of the corresponding nucleoside under stress conditions. The same is true for Gm, m5U and m5C modifications and corresponding methylation genes in different tissues during different developmental stages.ConclusionsWe identified candidate genes for various tRNA modified nucleosides in rice and Arabidopsis, especially on MTases for methylated nucleosides. Based on bioinformatics analysis, nucleoside abundance assessments and gene expression profiling, we propose four methylated nucleosides (Am, Cm, m1A and m7G) that are critical for stress response in rice and Arabidopsis, and three methylated nucleosides (Gm, m5U and m5C) that might be important during development.