Nucleosides In RNAs Research Articles

Modified nucleosides in eukaryotic messenger RNAs and their roles in developmental regulation

Epigenetic nucleoside modifications are critical for the stability and translational efficiency of messenger RNA. Depending on the organism, developmental stage, and tissue/organ investigated, the location and abundance of these nucleoside modifications may differ, which in turn influence the splicing event, half-life time of mature mRNA, as well as translation efficiency. Among the approximately 170 RNA nucleoside modifications, only a handful are found in mRNAs. The low abundance and high organ specificity make it a challenging work to study the role of each specific mRNA nucleoside modification. However, with the technical advances in recent years, including meRIP, great progress has been achieved, especially on the function of m⁶A and m⁵C epigenetic markers in eukaryotes. This review summarizes recent progress on nucleoside modifications of messenger RNAs, on their distribution on transcripts and their role in regulating growth anddevelopment. We also discuss the technical bottleneck and key issues for future investigation.

Application of solid-phase DNA probe method with cleavage by deoxyribozyme for analysis of long non-coding RNAs.

The solid-phase DNA probe method is a well-established technique for tRNA purification. We have applied this method for purification and analysis of other non-coding RNAs. Three columns for purification of tRNAPhe, transfer-messenger RNA (tmRNA) and 16S rRNA from Thermus thermophilus were connected in tandem and purifications were performed. From each column, tRNAPhe, tmRNA and 16S rRNA could be purified in a single step. This is the first report of purification of native tmRNA from T. thermophilus and the purification demonstrates that the solid-phase DNA probe method is applicable to non-coding RNA, which is present in lower amounts than tRNA. Furthermore, if a long non-coding RNA is cleaved site-specifically and the fragment can be purified by the solid-phase DNA probe method, modified nucleosides in the long non-coding RNA can be analysed. Therefore, we designed a deoxyribozyme (DNAzyme) to perform site-specific cleavage of 16S rRNA, examined optimum conditions and purified the resulting RNA fragment. Sequencing of complimentary DNA and mass spectrometric analysis revealed that the purified RNA corresponded to the targeted fragment of 16S rRNA. Thus, the combination of DNAzyme cleavage and purification using solid-phase DNA probe methodology can be a useful technique for analysis of modified nucleosides in long non-coding RNAs.

Using spectral matching to interpret LC-MS/MS data during RNA modification mapping.

While a number of approaches have been developed to analyze liquid chromatography tandem mass spectrometry (LC-MS/MS) data obtained from modified oligonucleotides, the majority of these methods require analyzing every MS/MS spectrum de novo to sequence the oligonucleotide and place the modification. Spectral matching is an alternative approach for analyzing MS/MS data that is based on creating a library of annotated MS/MS spectra against which individual MS/MS data can be searched. Here, we have adapted the existing NIST spectral matching software to enable its use in the interpretation of MS/MS data obtained from modified oligonucleotides. In particular, we demonstrate the utility of this approach to identify specific post-transcriptionally modified nucleosides in particular transfer RNAs (tRNAs) obtained through a conventional RNA modification mapping experimental protocol. Spectral matching was found to be an efficient approach for screening for known modified tRNAs by using the experimental data as the library and previously annotated RNase T1 digestion products of tRNAs as the reference spectra. The utility of spectral matching for rapid analysis of multiple LC-MS/MS analyses was demonstrated by screening mutant strains of Streptococcus mutans to identify the enzyme(s) responsible for synthesizing the tRNA position 37 modification threonylcarbamoyladenosine (t6 A).

Association of prenatal exposure to benzodiazepines and child internalizing problems: A sibling-controlled cohort study

Constitutive and regulated turnover of RNAs is necessary to eliminate aberrant RNA molecules and control the level of specific mRNAs to maintain homeostasis or to respond to signals in living cells. Modifications of nucleosides in specific RNAs are important in modulating the functions of these transcripts, but they can also dramatically impact their fate and turnover. This chapter will review how RNA modifications impact the activities of ribonucleases that target these RNAs for degradation or cleavage, focusing more particularly on tRNAs and mRNAs in eukaryotic cells. Many nucleoside modifications are important to promote proper folding of tRNAs, and the absence of specific modifications makes them susceptible to degradation by quality control pathways that eliminate improperly folded species. Modifications in tRNAs can also modulate their cleavage during stress or by fungal toxins that target modified nucleosides. Modifications of the cap structure found at the 5′-end of eukaryotic mRNAs are essential to control the degradation of these mRNAs. In addition, internal modifications of eukaryotic mRNAs can change their secondary structures or provide binding sites for reader proteins, which can dramatically impact their stability. Recent examples show that mRNA modifications play important roles in regulating mRNA stability during development, cellular differentiation and physiological responses. Finally, many modifications can impact microRNA- and siRNA-mediated gene regulation by direct or indirect effects. With the growing number of genomic techniques able to identify modifications genome wide, it is anticipated that novel chemical modifications or new modification sites will be identified, which will play additional regulatory functions for RNA turnover.

Method for producing high-concentration alcohol: Mitsubishi Chemical Corp, Mitsubishi Chemical Engineering Corp.

Constitutive and regulated turnover of RNAs is necessary to eliminate aberrant RNA molecules and control the level of specific mRNAs to maintain homeostasis or to respond to signals in living cells. Modifications of nucleosides in specific RNAs are important in modulating the functions of these transcripts, but they can also dramatically impact their fate and turnover. This chapter will review how RNA modifications impact the activities of ribonucleases that target these RNAs for degradation or cleavage, focusing more particularly on tRNAs and mRNAs in eukaryotic cells. Many nucleoside modifications are important to promote proper folding of tRNAs, and the absence of specific modifications makes them susceptible to degradation by quality control pathways that eliminate improperly folded species. Modifications in tRNAs can also modulate their cleavage during stress or by fungal toxins that target modified nucleosides. Modifications of the cap structure found at the 5′-end of eukaryotic mRNAs are essential to control the degradation of these mRNAs. In addition, internal modifications of eukaryotic mRNAs can change their secondary structures or provide binding sites for reader proteins, which can dramatically impact their stability. Recent examples show that mRNA modifications play important roles in regulating mRNA stability during development, cellular differentiation and physiological responses. Finally, many modifications can impact microRNA- and siRNA-mediated gene regulation by direct or indirect effects. With the growing number of genomic techniques able to identify modifications genome wide, it is anticipated that novel chemical modifications or new modification sites will be identified, which will play additional regulatory functions for RNA turnover.

Impact of RNA Modifications and RNA-Modifying Enzymes on Eukaryotic Ribonucleases.

Constitutive and regulated turnover of RNAs is necessary to eliminate aberrant RNA molecules and control the level of specific mRNAs to maintain homeostasis or to respond to signals in living cells. Modifications of nucleosides in specific RNAs are important in modulating the functions of these transcripts, but they can also dramatically impact their fate and turnover. This chapter will review how RNA modifications impact the activities of ribonucleases that target these RNAs for degradation or cleavage, focusing more particularly on tRNAs and mRNAs in eukaryotic cells. Many nucleoside modifications are important to promote proper folding of tRNAs, and the absence of specific modifications makes them susceptible to degradation by quality control pathways that eliminate improperly folded species. Modifications in tRNAs can also modulate their cleavage during stress or by fungal toxins that target modified nucleosides. Modifications of the cap structure found at the 5'-end of eukaryotic mRNAs are essential to control the degradation of these mRNAs. In addition, internal modifications of eukaryotic mRNAs can change their secondary structures or provide binding sites for reader proteins, which can dramatically impact their stability. Recent examples show that mRNA modifications play important roles in regulating mRNA stability during development, cellular differentiation and physiological responses. Finally, many modifications can impact microRNA- and siRNA-mediated gene regulation by direct or indirect effects. With the growing number of genomic techniques able to identify modifications genome wide, it is anticipated that novel chemical modifications or new modification sites will be identified, which will play additional regulatory functions for RNA turnover.

A High-Throughput Quantitative Approach Reveals More Small RNA Modifications in Mouse Liver and Their Correlation with Diabetes

Studies of RNA modification are usually focused on tRNA. However the modification of other small RNAs, including 5.8S rRNA, 5S rRNA, and small RNA sized at 10-60 nt, is still largely unknown. In this study, we established an efficient method based on liquid chromatography-tandem mass spectrometry (LC-MS/MS) to simultaneously identify and quantify more than 40 different types of nucleosides in small RNAs. With this method, we revealed 23 modified nucleosides of tRNA from mouse liver, and 6 of them were observed for the first time in eukaryotic tRNA. Moreover, 5 and 4 modified nucleosides were detected for the first time in eukaryotic 5.8S and 5S rRNA, respectively, and 22 modified nucleosides were identified in the small RNAs sized at 30-60 or 10-30 nt. Interestingly, two groups of 5S rRNA peaks were observed when analyzed by HPLC, and the abundance of modified nucleosides is significantly different between the two groups of peaks. Further studies show that multiple modifications in small RNA from diabetic mouse liver are significantly increased or decreased. Taken together, our data revealed more modified nucleosides in various small RNAs and showed the correlation of small RNA modifications with diabetes. These results provide new insights to the role of modifications of small RNAs in their stability, biological functions, and correlation with diseases.

3] Base-modified phosphoramidite analogs of pyrimidine ribonucleosides for RNA structure-activity studies

This chapter describes the design, synthesis, and utilization of base-modified analogs of natural ribonucleosides originally developed for the structure–activity studies of the hammerhead ribozyme. These compounds are also applied to study RNA–ligand interactions in the hairpin ribozyme, group II intron, VP vaccinia virus, and the development of a “subtraction mutagenesis” approach with basic modifications in the hammerhead system. The most frequently used method for the incorporation of modified nucleosides in synthetic RNAs is via solid-phase oligonucleotide synthesis based on phosphoramidite chemistry. This method utilizes the repetitive coupling of appropriately protected monomeric nucleotides to the growing oligonucleotide chain in the 3´ to 5 direction. After assembly of the chain is completed, removal of the phosphate, base, and 2´-OH protecting group results in target RNA with or without incorporated modified monomeric units. To incorporate a specifically modified nucleotide at a desired position, it is necessary to prepare a monomeric building block protected at the phosphate, base, and 2´-OH with protecting groups that are compatible with the general methodology of incorporation of regular unmodified monomers. Thus, synthesis of the target monomer includes the standard procedure for introduction of 5´-O-dimethoxytrityl group, 2´-O-tert-butyldimethylsilyl group and introduction of 3´-diisopropylaminocyanoethylphosphoramidite, and protection of the base.

Mapping to nucleotide resolution of pseudouridine residues in large subunit ribosomal RNAs from representative eukaryotes, prokaryotes, archaebacteria, mitochondria and chloroplasts

The pseudouridine (Ψ) residues present in the high molecular mass RNA from the large ribosomal subunit (LSU) have been sequenced from representative species of the eukaryotes, prokaryotes and archaebacteria, and from mitochondrial and chloroplast organelles. Ribosomes from Bacillus subtilis, Halobacter halobium, Drosphilia melanogaster, Mus musculus, Homo sapiens, mitochondria of M. musculus, H. sapiens andTrypanosoma brucei, and Zea mays chloroplasts were examined, resulting in the exact localization of 190 Ψ residues. The number of Ψ residues per RNA varied from one in the mitochondrial RNAs to 57 in the cytoplasmic LSU RNA ofD. melanogaster and M. musculus. Despite this, all of the Ψ residues were found in three domains, II, IV and V. All three are at or have been linked to the peptidyl transferase center according to the literature. Comparison of the sites for Ψ among the species examined revealed four conserved or semi-conserved segments. One is the region 1911 to 1917, which contains three Ψ or modified Ψ in almost all species examined. This site is also juxtaposed to the decoding site of the 30 S subunit in the 70 S ribosome and has been implicated in the fidelity of codon recognition. Three additional sites were at the peptidyl transferase center itself. The juxtaposition of the conserved sites for Ψ with the two important functions of the ribosome, codon recognition and peptide bond formation, implies an important role for Ψ in ribosome function.We report some new putative modified nucleosides in LSU RNAs as detected by reverse transcription, correct a segment of the sequence ofZ. mays chloroplasts and D. melanogaster LSU RNA, correlate the secondary structural context for all known Ψ residues in ribosomal RNA, and compare the sites for Ψ with those known for methylated nucleosides in H. sapiens.

Enzymatic conversion of adenosine to inosine and to N1-methylinosine in transfer RNAs: a review.

Inosine (6-deaminated adenosine) is a characteristic modified nucleoside that is found at the first anticodon position (position 34) of several tRNAs of eukaryotic and eubacterial origins, while N1-methylinosine is found exclusively at position 37 (3' adjacent to the anticodon) of eukaryotic tRNA(Ala) and at position 57 (in the middle of the psi loop) of several tRNAs from halophilic and thermophilic archaebacteria. Inosine has also been recently found in double-stranded RNA, mRNA and viral RNAs. As for all other modified nucleosides in RNAs, formation of inosine and inosine derivative in these RNA is catalysed by specific enzymes acting after transcription of the RNA genes. Using recombinant tRNAs and T7-runoff transcripts of several tRNA genes as substrates, we have studied the mechanism and specificity of tRNA-inosine-forming enzymes. The results show that inosine-34 and inosine-37 in tRNAs are both synthesised by a hydrolytic deamination-type reaction, catalysed by distinct tRNA:adenosine deaminases. Recognition of tRNA substrates by the deaminases does not strictly depend on a particular "identity' nucleotide. However, the efficiency of adenosine to inosine conversion depends on the nucleotides composition of the anticodon loop and the proximal stem as well as on 3D-architecture of the tRNA. In eukaryotic tRNA(Ala), N1-methylinosine-37 is formed from inosine-37 by a specific SAM-dependent methylase, while in the case of N1-methylinosine-57 in archaeal tRNAs, methylation of adenosine-57 into N1-methyladenosine-57 occurs before the deamination process. The T psi-branch of fragmented tRNA is the minimalist substrate for the N1-methylinosine-57 forming enzymes. Inosine-34 and N1-methylinosine-37 in human tRNA(Ala) are targets for specific autoantibodies which are present in the serum of patients with inflammatory muscle disease of the PL-12 polymyositis type. Here we discuss the mechanism, specificity and general properties of the recently discovered RNA:adenosine deaminases/editases acting on double-stranded RNA, intron-containing mRNA and viral RNA in relation to those of the deaminases acting on tRNAs.

Antisuppressor mutations and sulfur-carrying nucleosides in transfer RNAs of Schizosaccharomyces pombe.

Antisuppressor mutations reduce the efficiency of nonsense suppressors. A mutation in the gene sin4 of Schizosaccharomyces pombe leads to loss of 5-(methoxycarbonylmethyl) thiouridine (mcm5s2U) from the first anticodon position of tRNAs. This resembles the phenotype of sin3 (Heyer, W. D., Thuriaux, P., Kohli, J., Ebert, P., Kersten, H., Gehrke, C., Kuo, K. C., and Agris, P. F. (1984) J. Biol. Chem. 259, 2856-2862), but the mutations reside in different genes. In vivo 35S-labeled tRNA from the parental suppressor strain sup3, the antisuppressor strains sin3 and sin4, and the double mutant sin3 sin4 has been digested to nucleosides and analyzed with high performance liquid chromatography methods. The major sulfur-carrying nucleoside in wild-type S. pombe tRNA is mcm5s2U. It is reduced in the mutant strains. Two other thiolated nucleosides are also present: 2-thiouridine and a nucleoside of unknown structure. Neither was affected by the antisuppressor mutations. Thiocytidine has not been found. Independent from their effect on suppressors, the two mutations sin3 and sin4 reduce the growth rate of cells, and sin3 also increases cell length. In vivo decoding of the serine codon UCG by the UCA reading serine tRNA is not promoted by the two antisuppressor mutations.