Nucleoside Modifications Research Articles

MRNA: A Novel Avenue to Antibody Therapy?

First attempts to use exogenous mRNA for protein expression invivo were made more than 25 years ago. However, widespread appreciation of invitro transcribed mRNA as a powerful technology for supplying therapeutic proteins to the body has evolved only during the past few years. Various approaches to turning mRNA into a potent therapeutic have been developed. All of them share utilization of specifically designed, rather than endogenous, sequences and thorough purification protocols. Apart from this, there are two fundamental philosophies, one promoting the use of chemically modified nucleotides, the other advocating restriction to unmodified building blocks. Meanwhile, both strategies have received broad support by successful mRNA-based protein treatments in animal models. For such invivo use, specifically optimized mRNA had to be combined with potent formulations to enable efficient invivo delivery. The present review analyzes the applicability of mRNA technology to antibody therapy in all main fields: antitoxins, infectious diseases, and oncology.

Analysis of the Cellular Roles of MOCS3 Identifies a MOCS3-Independent Localization of NFS1 at the Tips of the Centrosome.

The deficiency of the molybdenum cofactor (Moco) is an autosomal recessive disease, which leads to the loss of activity of all molybdoenzymes in humans with sulfite oxidase being the essential protein. Moco deficiency generally results in death in early childhood. Moco is a sulfur-containing cofactor synthesized in the cytosol with the sulfur being provided by a sulfur relay system composed of the l-cysteine desulfurase NFS1, MOCS3, and MOCS2A. Human MOCS3 is a dual-function protein that was shown to play an important role in Moco biosynthesis and in the mcm5s2U thio modifications of nucleosides in cytosolic tRNAs for Lys, Gln, and Glu. In this study, we constructed a homozygous MOCS3 knockout in HEK293T cells using the CRISPR/Cas9 system. The effects caused by the absence of MOCS3 were analyzed in detail. We show that sulfite oxidase activity was almost completely abolished, on the basis of the absence of Moco in these cells. In addition, mcm5s2U thio-modified tRNAs were not detectable. Because the l-cysteine desulfurase NFS1 was shown to act as a sulfur donor for MOCS3 in the cytosol, we additionally investigated the impact of a MOCS3 knockout on the cellular localization of NFS1. By different methods, we identified a MOCS3-independent novel localization of NFS1 at the centrosome.

TRNA Processing and Subcellular Trafficking Proteins Multitask in Pathways for Other RNAs.

This article focuses upon gene products that are involved in tRNA biology, with particular emphasis upon post-transcriptional RNA processing and nuclear-cytoplasmic subcellular trafficking. Rather than analyzing these proteins solely from a tRNA perspective, we explore the many overlapping functions of the processing enzymes and proteins involved in subcellular traffic. Remarkably, there are numerous examples of conserved gene products and RNP complexes involved in tRNA biology that multitask in a similar fashion in the production and/or subcellular trafficking of other RNAs, including small structured RNAs such as snRNA, snoRNA, 5S RNA, telomerase RNA, and SRP RNA as well as larger unstructured RNAs such as mRNAs and RNA-protein complexes such as ribosomes. Here, we provide examples of steps in tRNA biology that are shared with other RNAs including those catalyzed by enzymes functioning in 5′ end-processing, pseudoU nucleoside modification, and intron splicing as well as steps regulated by proteins functioning in subcellular trafficking. Such multitasking highlights the clever mechanisms cells employ for maximizing their genomes.

Purification of mRNA Encoding Chimeric Antigen Receptor Is Critical for Generation of a Robust T-Cell Response.

T cells made with messenger RNA (mRNA) encoding chimeric antigen receptor (CAR) offer a safe alternative to those transduced with viral CARs by mitigating the side effects of constitutively active T cells. Previous studies have shown that mRNA CAR T cells are transiently effective but lack persistence and potency across tumor types. It was hypothesized that the efficacy of mRNA CARs could be improved by utilizing recent advancements in RNA technology, such as incorporating a modified nucleoside, 1-methylpseudouridine, into the mRNA and applying a novel purification method using RNase III to eliminate dsRNA contaminants. T cells electroporated with nucleoside-modified and purified mRNA encoding CD19 CAR showed an initial twofold increase in CAR surface expression, as well as a twofold improvement in cytotoxic killing of leukemia cells that persisted up to 5 days. T cells generated with nucleoside-modified and purified CAR mRNA also showed reduced expression of checkpoint regulators and a differential pattern of genetic activation compared to those made with conventional mRNA. In vivo studies using a leukemia mouse model revealed that the most robust 100-fold suppression of leukemic burden was achieved using T cells electroporated with purified mRNAs, regardless of their nucleoside modification. The results provide a novel approach to generate mRNA for clinical trials, and poise mRNA CAR T cells for increased efficacy during testing as new CAR targets emerge.

Gas-phase structures of protonated arabino nucleosides

Nucleoside modification plays an important role in the native function of DNA and RNA and is also an important synthetic tool for pharmaceuticals. The gas-phase structures of several protonated arabino nucleosides, an important family of modified nucleoside pharmaceuticals, are examined in this work. Infrared multiple photon dissociation action spectra are collected for the protonated forms of the adenine, cytosine, guanine, and uracil arabinosides ([araAdo+H]+, [araCyd+H]+, [araGuo+H]+, and [araUrd+H]+) in the IR fingerprint and hydrogen-stretching regions. Electronic structure calculations are performed to determine low-energy conformers of [araAdo+H]+, [araCyd+H]+, [araGuo+H]+, and [araUrd+H]+, and generate predicted IR spectra for comparison to experiments. Conformers displaying a unique O2′H⋯O5′ hydrogen-bonding interaction are populated in each of these systems. Conformers exhibiting hydrogen-bonding interactions between the nucleobase and O5′ are also found to display good agreement with the measured spectra. Competition between sugar–sugar, and nucleobase–sugar hydrogen bonding reveals a preference for [araCyd+H]+, [araGuo+H]+, and [araUrd+H]+ to stabilize the sugar ring pucker over the nucleobase rotation. N3 protonation of [araAdo+H]+ provides a very strong N3H+⋯O5′ hydrogen-bonding interaction, such that nucleobase–sugar hydrogen-bonding takes energetic preference over stabilization of the sugar puckering. However, conformers exhibiting each mode of hydrogen bonding contribute to the measured spectrum of [araAdo+H]+.

Production and Application of Stable Isotope-Labeled Internal Standards for RNA Modification Analysis.

Post-transcriptional RNA modifications have been found to be present in a wide variety of organisms and in different types of RNA. Nucleoside modifications are interesting due to their already known roles in translation fidelity, enzyme recognition, disease progression, and RNA stability. In addition, the abundance of modified nucleosides fluctuates based on growth phase, external stress, or possibly other factors not yet explored. With modifications ever changing, a method to determine absolute quantities for multiple nucleoside modifications is required. Here, we report metabolic isotope labeling to produce isotopically labeled internal standards in bacteria and yeast. These can be used for the quantification of 26 different modified nucleosides. We explain in detail how these internal standards are produced and show their mass spectrometric characterization. We apply our internal standards and quantify the modification content of transfer RNA (tRNA) from bacteria and various eukaryotes. We can show that the origin of the internal standard has no impact on the quantification result. Furthermore, we use our internal standard for the quantification of modified nucleosides in mouse tissue messenger RNA (mRNA), where we find different modification profiles in liver and brain tissue.

Guide snoRNAs: Drivers or Passengers in Human Disease?

In every domain of life, RNA-protein interactions play a significant role in co- and post-transcriptional modifications and mRNA translation. RNA performs diverse roles inside the cell, and therefore any aberrancy in their function can cause various diseases. During maturation from its primary transcript, RNA undergoes several functionally important post-transcriptional modifications including pseudouridylation and ribose 2′-O-methylation. These modifications play a critical role in the stability of the RNA. In the last few decades, small nucleolar RNAs (snoRNAs) were revealed to be one of the main components to guide these modifications. Due to their active links to the nucleoside modification, deregulation in the snoRNA expressions can cause multiple disorders in humans. Additionally, host genes carrying snoRNA-encoding sequences in their introns also show differential expression in disease. Although few reports support a causal link between snoRNA expression and disease manifestation, this emerging field will have an impact on the way we think about biomarkers or identify novel targets for therapy. This review focuses on the intriguing aspect of snoRNAs that function as a guide in post-transcriptional RNA modification, and regulation of their host genes in human disease.

Structural insight into antisense gapmer-RNA oligomer duplexes through molecular dynamics simulations

There is an extensive research carrying out on antisense technology and the molecules entering into clinical trials are increasing rapidly. Phosphorothioate (PS) is a chemical modification in which nonbridged oxygen is replaced with a sulfur, consequently providing resistance against nuclease activity. The 2'-4' conformationally restricted nucleoside has the structural features of both 2'-O-methoxy ethyl RNA (MOE), which shows good toxicity profile, and locked nucleic acid (LNA), which shows good binding affinity towards the target RNA. These modifications have been studied and suggested that they can be a potential therapeutic agents in antisense therapy. Mipomersen (ISIS 301012), which contains the novel nucleoside modification has been used to target to apolipoprotein (Apo B), which reduces LDL cholesterol by 6–41%. In this study, classical molecular dynamics (MD) simulations were performed on six different antisense gapmer/target-RNA oligomer duplexes (LNA-PS-LNA/RNA, RcMOE-PS-RcMOE/RNA, ScMOE-PS-ScMOE/RNA, MOE-PS-MOE/RNA, PS-DNA/RNA and DNA/RNA) to investigate the structural dynamics, stability and solvation properties. The LNA, MOE nucleotides present in respective duplexes are showing the structure of A-form and the PS-DNA nucleotides resemble the structure of B-form helix with respect to some of the helical parameters. Free energy calculations suggest that the oligomer, which contains LNA binds to the RNA strongly than other modifications as shown in experimental results. The MOE modified nucleotide, which although had a lower binding affinity but higher solvent accessible surface area (SASA) compared to the other modifications, may be influencing the toxicity and hence may be used it in Mipomersen, the second antisense molecule which is approved by FDA.Communicated by Ramaswamy H. Sarma

Nano LC-MS using capillary columns enables accurate quantification of modified ribonucleosides at low femtomol levels.

Post-transcriptional chemical modifications of (t)RNA molecules are crucial in fundamental biological processes, such as translation. Despite their biological importance and accumulating evidence linking them to various human diseases, technical challenges have limited their detection and accurate quantification. Here, we present a sensitive capillary nanoflow liquid chromatography mass spectrometry (nLC-MS) pipeline for quantitative high-resolution analysis of ribonucleoside modifications from complex biological samples. We evaluated two porous graphitic carbon (PGC) materials and one end-capped C18 reference material as stationary phases for reversed-phase separation. We found that these matrices have complementing retention and separation characteristics, including the capability to separate structural isomers. PGC and C18 matrices yielded excellent signal-to-noise ratios in nLC-MS while differing in the separation capability and sensitivity for various nucleosides. This emphasizes the need for tailored LC-MS setups for optimally detecting as many nucleoside modifications as possible. Detection ranges spanning up to six orders of magnitude enable the analysis of individual ribonucleosides down to femtomol concentrations. Furthermore, normalizing the obtained signal intensities to a stable isotope labeled spike-in enabled direct comparison of ribonucleoside levels between different samples. In conclusion, capillary columns coupled to nLC-MS constitute a powerful and sensitive tool for quantitative analysis of modified ribonucleosides in complex biological samples. This setup will be invaluable for further unraveling the intriguing and multifaceted biological roles of RNA modifications.

Existence of Diverse Modifications in Small-RNA Species Composed of 16-28 Nucleotides.

RNA contains diverse modifications that exert an important influence in a variety of cellular processes. So far, more than 150 modifications have been identified in various RNA species, mainly in ribosomal RNA (rRNA), transfer RNA (tRNA), and messenger RNA (mRNA). In contrast to rRNA, tRNA, and mRNA, the known modifications in small RNA species have been primarily limited to 2'-O-ribose methylation in plants and inosine in mammals. The methylation of small RNAs in mammals is still unclear. Current methods widely used in the characterization of small RNAs are mainly based on the strategy of nucleic acid hybridization and sequencing, which cannot characterize modifications in small RNAs. Herein, we have systematically investigated modifications in small RNAs composed of 16-28 nucleotides (nt) by establishing an effective isolation and neutral enzymatic digestion of small RNAs in combination with liquid chromatography/electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS). This method allowed us to simultaneously detect 57 different types of nucleoside modification. By using this approach, we revealed 24 modifications in small RNAs comprising 16-28 nt from human cells. In addition, we found that the obesity-associated protein (FTO) may demethylate N6 -methyladenosine (m6 A) and N6 ,2'-O-dimethyladenosine (m6 Am) in small RNAs of 16-28 nt. Our study demonstrates the existence of diverse modifications in small RNAs composed of 16-28 nt, which may promote in-depth understanding of the regulatory roles of noncoding RNAs.

Differentiating Positional Isomers of Nucleoside Modifications by Higher-Energy Collisional Dissociation Mass Spectrometry (HCD MS).

The analytical identification of positional isomers (e.g., 3-, N4-, 5-methylcytidine) within the > 160 different post-transcriptional modifications found in RNA can be challenging. Conventional liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) approaches rely on chromatographic separation for accurate identification because the collision-induced dissociation (CID) mass spectra of these isomers nearly exclusively yield identical nucleobase ions (BH2+) from the same molecular ion (MH+). Here, we have explored higher-energy collisional dissociation (HCD) as an alternative fragmentation technique to generate more informative product ions that can be used to differentiate positional isomers. LC-MS/MS of modified nucleosides characterized using HCD led to the creation of structure- and HCD energy-specific fragmentation patterns that generated unique fingerprints, which can be used to identify individual positional isomers even when they cannot be separated chromatographically. While particularly useful for identifying positional isomers, the fingerprinting capabilities enabled by HCD also offer the potential to generate HPLC-independent spectral libraries for the rapid analysis of modified ribonucleosides. Graphical Abstract ᅟ.

TRNA modification profiles of non-small cell lung cancer (NSCLC).

e20542Background: Transfer RNAs (tRNAs) are key molecules participating in protein synthesis. Modifications of nucleosides on tRNA are crucial for efficient and accurate protein synthesis, and modi...

Avoiding Antibiotic Inactivation in Mycobacterium tuberculosis by Rv3406 through Strategic Nucleoside Modification.

5'-[ N-(d-biotinoyl)sulfamoyl]amino-5'-deoxyadenosine (Bio-AMS, 1) possesses selective activity against Mycobacterium tuberculosis ( Mtb) and arrests fatty acid and lipid biosynthesis through inhibition of the Mycobacterium tuberculosis biotin protein ligase ( MtBPL). Mtb develops spontaneous resistance to 1 with a frequency of at least 1 × 10-7 by overexpression of Rv3406, a type II sulfatase that enzymatically inactivates 1. In an effort to circumvent this resistance mechanism, we describe herein strategic modification of the nucleoside at the 5'-position to prevent enzymatic inactivation. The new analogues retained subnanomolar potency to MtBPL ( KD = 0.66-0.97 nM), and 5' R- C-methyl derivative 6 exhibited identical antimycobacterial activity toward: Mtb H37Rv, MtBPL overexpression, and an isogenic Rv3406 overexpression strain (minimum inhibitory concentration, MIC = 1.56 μM). Moreover, 6 was not metabolized by recombinant Rv3406 and resistant mutants to 6 could not be isolated (frequency of resistance <1.4 × 10-10) demonstrating it successfully overcame Rv3406-mediated resistance.

Oxidatively-induced DNA damage and base excision repair in euthymic patients with bipolar disorder

Oxidatively-induced DNA damage has previously been associated with bipolar disorder. More recently, impairments in DNA repair mechanisms have also been reported. We aimed to investigate oxidatively-induced DNA lesions and expression of DNA glycosylases involved in base excision repair in euthymic patients with bipolar disorder compared to healthy individuals. DNA base lesions including both base and nucleoside modifications were measured using gas chromatography-tandem mass spectrometry and liquid chromatography-tandem mass spectrometry with isotope-dilution in DNA samples isolated from leukocytes of euthymic patients with bipolar disorder (n = 32) and healthy individuals (n = 51). The expression of DNA repair enzymes OGG1 and NEIL1 were measured using quantitative real-time polymerase chain reaction. The levels of malondialdehyde were measured using high performance liquid chromatography. Seven DNA base lesions in DNA of leukocytes of patients and healthy individuals were identified and quantified. Three of them had significantly elevated levels in bipolar patients when compared to healthy individuals. No elevation of lipid peroxidation marker malondialdehyde was observed. The level of OGG1 expression was significantly reduced in bipolar patients compared to healthy individuals, whereas the two groups exhibited similar levels of NEIL1 expression. Our results suggest that oxidatively-induced DNA damage occurs and base excision repair capacity may be decreased in bipolar patients when compared to healthy individuals. Measurement of oxidatively-induced DNA base lesions and the expression of DNA repair enzymes may be of great importance for large scale basic research and clinical studies of bipolar disorder.

Role of RNA modifications in brain and behavior.

Much progress in our understanding of RNA metabolism has been made since the first RNA nucleoside modification was identified in 1957. Many of these modifications are found in noncoding RNAs but recent interest has focused on coding RNAs. Here, we summarize current knowledge of cellular consequences of RNA modifications, with a special emphasis on neuropsychiatric disorders. We present evidence for the existence of an "RNA code," similar to the histone code, that fine-tunes gene expression in the nervous system by using combinations of different RNA modifications. Unlike the relatively stable genetic code, this combinatorial RNA epigenetic code, or epitranscriptome, may be dynamically reprogrammed as a cause or consequence of psychiatric disorders. We discuss potential mechanisms linking disregulation of the epitranscriptome with brain disorders and identify potential new avenues of research.

Heavy Chronic Intermittent Ethanol Exposure Alters Small Noncoding RNAs in Mouse Sperm and Epididymosomes

While the risks of maternal alcohol abuse during pregnancy are well-established, several preclinical studies suggest that chronic preconception alcohol consumption by either parent may also have significance consequences for offspring health and development. Notably, since isogenic male mice used in these studies are not involved in gestation or rearing of offspring, the cross-generational effects of paternal alcohol exposure suggest a germline-based epigenetic mechanism. Many recent studies have demonstrated that the effects of paternal environmental exposures such as stress or malnutrition can be transmitted to the next generation via alterations to small noncoding RNAs in sperm. Therefore, we used high throughput sequencing to examine the effect of preconception ethanol on small noncoding RNAs in sperm. We found that chronic intermittent ethanol exposure altered several small noncoding RNAs from three of the major small RNA classes in sperm, tRNA-derived small RNA (tDR), mitochondrial small RNA, and microRNA. Six of the ethanol-responsive small noncoding RNAs were evaluated with RT-qPCR on a separate cohort of mice and five of the six were confirmed to be altered by chronic ethanol exposure, supporting the validity of the sequencing results. In addition to altered sperm RNA abundance, chronic ethanol exposure affected post-transcriptional modifications to sperm small noncoding RNAs, increasing two nucleoside modifications previously identified in mitochondrial tRNA. Furthermore, we found that chronic ethanol reduced epididymal expression of a tRNA methyltransferase, Nsun2, known to directly regulate tDR biogenesis. Finally, ethanol-responsive sperm tDR are similarly altered in extracellular vesicles of the epididymis (i.e., epididymosomes), supporting the hypothesis that alterations to sperm tDR emerge in the epididymis and that epididymosomes are the primary source of small noncoding RNAs in sperm. These results add chronic ethanol to the growing list of paternal exposures that can affect small noncoding RNA abundance and nucleoside modifications in sperm. As small noncoding RNAs in sperm have been shown to causally induce heritable phenotypes in offspring, additional research is warranted to understand the potential effects of ethanol-responsive sperm small noncoding RNAs on offspring health and development.

The Effects of Ultraviolet Radiation on Nucleoside Modifications in RNA.

Ultraviolet radiation (UVR) is a known genotoxic agent. Although its effects on DNA have been well-documented, its impact on RNA and RNA modifications is less studied. By using Escherichia coli tRNA (tRNA) as a model system, we identify the UVA (370 nm) susceptible chemical groups and bonds in a large variety of modified nucleosides. We use liquid chromatography tandem mass spectrometry to identify specific nucleoside photoproducts under in vitro and in vivo conditions, which were then verified by employing stable-isotope labeled tRNAs. These studies suggest that the -amino or -oxy groups of modified nucleosides, in addition to sulfur, are labile in the oxidative environment generated by UVA exposure. Further, these studies document a range of RNA photoproducts and post-transcriptional modifications that arise because of UVR-induced cellular stress.

Profiling of modified nucleosides from ribonucleic acid digestion by supercritical fluid chromatography coupled to high resolution mass spectrometry

A method based on supercritical fluid chromatography coupled to high resolution mass spectrometry for the profiling of canonical and modified nucleosides was optimized, and compared to classical reverse-phase liquid chromatography in terms of separation, number of detected modified nucleosides and sensitivity. Limits of detection and quantification were measured using statistical method and quantifications of twelve nucleosides of a tRNA digest from E. coli are in good agreement with previously reported data. Results highlight the complementarity of both separation techniques to cover the largest view of nucleoside modifications for forthcoming epigenetic studies.

The fungal snoRNAome.

Small nucleolar RNAs (snoRNAs) are essential players in the rRNA biogenesis due to their involvement in the nucleolytic processing of the precursor and the subsequent guidance of nucleoside modifications. Within the kingdom Fungi, merely a few species-specific surveys have explored their snoRNA repertoire. However, the wide range of the snoRNA landscape spanning all major fungal lineages has not been mapped so far, mainly because of missing tools for automatized snoRNA detection and functional analysis. For the first time, we report here a comprehensive inventory of fungal snoRNAs together with a functional analysis and an in-depth investigation of their evolutionary history including innovations, deletions, and target switches. This large-scale analysis, incorporating more than 120 snoRNA families with more than 7700 individual snoRNA sequences, catalogs and clarifies the landscape of fungal snoRNA families, assigns functions to previously orphan snoRNAs, and increases the number of sequences by 450%. We also show that the snoRNAome is subject to ongoing rearrangements and adaptations, e.g., through lineage-specific targets and redundant guiding functions.

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.