tRNA Components Research Articles

Small RNA Profiles of Brain Tissue-Derived Extracellular Vesicles in Alzheimer's Disease.

Extracellular vesicles (EVs) and non-coding RNAs (ncRNAs) are emerging contributors to Alzheimer's disease (AD) pathophysiology. Differential abundance of ncRNAs carried by EVs may provide valuable insights into underlying disease mechanisms. Brain tissue-derived EVs (bdEVs) are particularly relevant, as they may offer valuable insights about the tissue of origin. However, there is limited research on diverse ncRNA species in bdEVs in AD. This study explored whether the non-coding RNA composition of EVs isolated from post-mortem brain tissue is related to AD pathogenesis. bdEVs from age-matched late-stage AD patients (n = 23) and controls (n = 10) that had been separated and characterized in our previous study were used for RNA extraction, small RNA sequencing, and qPCR verification. Significant differences of non-coding RNAs between AD and controls were found, especially for miRNAs and tRNAs. AD pathology-related miRNA and tRNA differences of bdEVs partially matched expression differences in source brain tissues. AD pathology had a more prominent association than biological sex with bdEV miRNA and tRNA components in late-stage AD brains. Our study provides further evidence that EV non-coding RNAs from human brain tissue, including but not limited to miRNAs, may be altered and contribute to AD pathogenesis.

2-Selenouridine, a Modified Nucleoside of Bacterial tRNAs, Its Reactivity in the Presence of Oxidizing and Reducing Reagents.

The 5-substituted 2-selenouridines are natural components of the bacterial tRNA epitranscriptome. Because selenium-containing biomolecules are redox-active entities, the oxidation susceptibility of 2-selenouridine (Se2U) was studied in the presence of hydrogen peroxide under various conditions and compared with previously reported data for 2-thiouridine (S2U). It was found that Se2U is more susceptible to oxidation and converted in the first step to the corresponding diselenide (Se2U)2, an unstable intermediate that decomposes to uridine and selenium. The reversibility of the oxidized state of Se2U was demonstrated by the efficient reduction of (Se2U)2 to Se2U in the presence of common reducing agents. Thus, the 2-selenouridine component of tRNA may have antioxidant potential in cells because of its ability to react with both cellular ROS components and reducing agents. Interestingly, in the course of the reactions studied, we found that (Se2U)2 reacts with Se2U to form new ‘oligomeric nucleosides′ as linear and cyclic byproducts.

Codon-Reduced Protein Synthesis With Manipulating tRNA Components in Cell-Free System.

Manipulating transfer RNAs (tRNAs) for emancipating sense codons to simplify genetic codons in a cell-free protein synthesis (CFPS) system can offer more flexibility and controllability. Here, we provide an overview of the tRNA complement protein synthesis system construction in the tRNA-depleted Protein synthesis Using purified Recombinant Elements (PURE) system or S30 extract. These designed polypeptide coding sequences reduce the genetic codon and contain only a single tRNA corresponding to a single amino acid in this presented system. Strategies for removing tRNAs from cell lysates and synthesizing tRNAs in vivo/vitro are summarized and discussed in detail. Furthermore, we point out the trend toward a minimized genetic codon for reducing codon redundancy by manipulating tRNAs in the different proteins. It is hoped that the tRNA complement protein synthesis system can facilitate the construction of minimal cells and expand the biomedical application scope of synthetic biology.

D-Amino Acid-Mediated Translation Arrest Is Modulated by the Identity of the Incoming Aminoacyl-tRNA.

A complete understanding of the determinants that restrict d-amino acid incorporation by the ribosome, which is of interest to both basic biologists and the protein engineering community, remains elusive. Previously, we demonstrated that d-amino acids are successfully incorporated into the C-terminus of the nascent polypeptide chain. Ribosomes carrying the resulting peptidyl-d-aminoacyl-tRNA (peptidyl-d-aa-tRNA) donor substrate, however, partition into subpopulations that either undergo translation arrest through inactivation of the ribosomal peptidyl-transferase center (PTC) or remain translationally competent. The proportion of each subpopulation is determined by the identity of the d-amino acid side chain. Here, we demonstrate that the identity of the aminoacyl-tRNA (aa-tRNA) acceptor substrate that is delivered to ribosomes carrying a peptidyl-d-aa-tRNA donor further modulates this partitioning. Our discovery demonstrates that it is the pairing of the peptidyl-d-aa-tRNA donor and the aa-tRNA acceptor that determines the activity of the PTC. Moreover, we provide evidence that both the amino acid and tRNA components of the aa-tRNAacceptorcontribute synergistically to the extent of arrest. The results of this work deepen our understanding of the mechanism of d-amino acid-mediated translation arrest and how cells avoid this precarious obstacle, reveal similarities to other translation arrest mechanisms involving the PTC, and provide a new route for improving the yields of engineered proteins containing d-amino acids.

Search of multiple hot spots on the surface of peptidyl-tRNA hydrolase: structural, binding and antibacterial studies.

Peptidyl-tRNA hydrolase (Pth) catalyzes the breakdown of peptidyl-tRNA into peptide and tRNA components. Pth from Acinetobacter baumannii (AbPth) was cloned, expressed, purified and crystallized in a native unbound (AbPth-N) state and in a bound state with the phosphate ion and cytosine arabinoside (cytarabine) (AbPth-C). Structures of AbPth-N and AbPth-C were determined at 1.36 and 1.10 Å resolutions, respectively. The structure of AbPth-N showed that the active site is filled with water molecules. In the structure of AbPth-C, a phosphate ion is present in the active site, while cytarabine is bound in a cleft which is located away from the catalytic site. The cytarabine-binding site is formed with residues: Gln19, Trp27, Glu30, Gln31, Lys152, Gln158 and Asp162. In the structure of AbPth-N, the side chains of two active-site residues, Asn70 and Asn116, were observed in two conformations. Upon binding of the phosphate ion in the active site, the side chains of both residues were ordered to single conformations. Since Trp27 is present at the cytarabine-binding site, the fluorescence studies were carried out which gave a dissociation constant (KD) of 3.3 ± 0.8 × 10-7 M for cytarabine. The binding studies using surface plasmon resonance gave a KD value of 3.7 ± 0.7 × 10-7 M. The bacterial inhibition studies using the agar diffusion method and the biofilm inhibition assay established the strong antimicrobial potential of cytarabine. It also indicated that cytarabine inhibited Gram-negative bacteria more profoundly when compared with Gram-positive bacteria in a dose-dependent manner. Cytarabine was also effective against the drug-resistant bacteria both alone as well as in combination with other antibiotics.

Gene control using tRNA‐shRNA chimeras: knockdown by shRNA and degradation of tRNA (749.3)

Gene therapy using RNA interference (RNAi) mediated by short hairpin RNAs (shRNAs) targeted against HIV‐1 is a promising alternative to antiretroviral therapy. Fusing shRNA to carrier tRNA to produce a tRNA‐shRNA chimera can provide a tunable level of shRNA delivery that is dependent on the action of the enzyme tRNaseZ to separate the shRNA and tRNA components of the chimera. We have introduced changes in the sequence of the acceptor stem of the tRNA portion of the tRNA‐shRNA chimera to produce the next‐generation of tRNA‐shRNA chimeras. Our targeted mutations are designed to result in an isomerization that triggers rapid tRNA decay after release of the shRNA by tRNaseZ. The knockdown efficiency of the resulting constructs can be assayed by a dual luciferase assay.Grant Funding Source: Supported by NSF CHE‐1005253

Mt-tRNA Components: Synthesis of (2-Thio)Uridines Modified with Blocked Glycine/Taurine Moieties at C-5,1

In this paper, we discuss the usefulness of reductive amination of 5-formyl-2′,3′-O-isopropylidene(-2-thio)uridine with glycine or taurine esters in the presence of sodium triacetoxyborohydride (NaBH(OAc)3) for the synthesis of the native mitochondrial (mt) tRNA components 5-carboxymethylaminomethyl(-2-thio)uridine (cmnm5(s2)U) and 5-taurinomethyl(-2-thio)uridine (τm5(s2)U) with a blocked amino acid function. 2-(Trimethylsilyl)ethyl and 2-(p-nitrophenyl)ethyl esters of glycine and 2-(2,4,5-trifluorophenyl)ethyl ester of taurine were selected as protection of carboxylic and sulfonic acid residues, respectively. The first synthesis of 5-formyl-2′,3′-O-isopropylidene-2-thiouridine is also reported.

The Origin and Evolution of tRNA Inferred from Phylogenetic Analysis of Structure

The evolutionary history of the two structural and functional domains of tRNA is controversial but harbors the secrets of early translation and the genetic code. To explore the origin and evolution of tRNA, we reconstructed phylogenetic trees directly from molecular structure. Forty-two structural characters describing the geometry of 571 tRNAs and three statistical parameters describing thermodynamic and mechanical features of molecules quantitatively were used to derive phylogenetic trees of molecules and molecular substructures. Trees of molecules failed to group tRNA according to amino acid specificity and did not reveal the tripartite nature of life, probably due to loss of phylogenetic signal or because tRNA diversification predated organismal diversification. Trees of substructures derived from both structural and statistical characters support the origin of tRNA in the acceptor arm and the hypothesis that the top half domain composed of acceptor and pseudouridine (TPsiC) arms is more ancient than the bottom half domain composed of dihydrouridine (DHU) and anticodon arms. This constitutes the cornerstone of the genomic tag hypothesis that postulates tRNAs were ancient telomeres in the RNA world. The trees of substructures suggest a model for the evolution of the major functional and structural components of tRNA. In this model, short RNA hairpins with stems homologous to the acceptor arm of present day tRNAs were extended with regions homologous to TPsiC and anticodon arms. The DHU arm was then incorporated into the resulting three-stemmed structure to form a proto-cloverleaf structure. The variable region was the last structural addition to the molecular repertoire of evolving tRNA substructures.

Synthesis of 2′‐O‐β‐d‐Ribofuranosylnucleosides

AbstractA simple and efficient method for the preparation of 2‐O‐β‐D‐ribofuranosylnucleosides, minor tRNA components, is described in this unit. The method consists of condensation of a small excess of 1‐O‐acetyl‐2,3,5‐tri‐O‐benzoyl‐β‐D‐ribofuranose activated with tin tetrachloride with N‐protected 3,5‐O‐tetra‐isopropyldisiloxane‐1,3‐diyl‐ribonucleosides in 1,2‐dichloroethane. Subsequent deprotection produces 2‐O‐β‐D‐ribofuranosylnucleosides in an overall yield of 46% to 72%.

Synthesis of RNA ContainingO-β-D-Ribofuranosyl-(1″2′)-adenosine-5″-phosphate and 1-Methyladenosine, Minor Components of tRNA

tRNA is best known for its function as amino acid carrier in the translation process, using the anticodon loop in the recognition process with mRNA. However, the impact of tRNA on cell function is much wider, and mutations in tRNA can lead to a broad range of diseases. Although the cloverleaf structure of tRNA is well-known based on X-ray-diffraction studies, little is known about the dynamics of this fold, the way structural dynamics of tRNA is influenced by the modified nucleotides present in tRNA, and their influence on the recognition of tRNA by synthetases, ribosomes, and other biomolecules. One of the reasons for this is the lack of good synthetic methods to incorporate modified nucleotides in tRNA so that larger amounts become available for NMR studies. Except of 2'-O-methylated nucleosides, only one other sugar-modified nucleoside is present in tRNA, i.e., 2'-O-beta-D-ribofuranosyl nucleosides. The T loop of tRNA often contains charged modified nucleosides, of which 1-methyladenosine and phosphorylated disaccharide nucleosides are striking examples. A protecting-group strategy was developed to introduce 1-methyladenosine and 5''-O-phosphorylated 2'-O-(beta-D-ribofuranosyl)-beta-D-ribofuranosyladenine in the same RNA fragment. The phosphorylation of the disaccharide nucleoside was performed after the assembly of the RNA on solid support. The modified RNA was characterized by mass-spectrometry analysis from the RNase T1 digestion fragments. The successful synthesis of this T loop of the tRNA of Schizosaccharomyces pombe initiator tRNA(Met) will be followed by its structural analysis by NMR and by studies on the influence of these modified nucleotides on dynamic interactions within the complete tRNA.

Efficient and faithful in vitro translation of natural and synthetic mRNA with human ribosomes

Efficient systems for in vitro translation are of importance for biochemical and gene expression studies as well as for biotechnological developments. We optimized a cell-free translation system using subcellular fractions from human placenta and high quality placental tRNAs isolated using a simple and fast procedure. The postmitochondrial fraction or a reconstituted system containing soluble proteins plus polysomes were able to efficiently translate endogenous and exogenous mRNAs. Optima for ions, enzymes, tRNA and energy mix components were determined for a poly(U)-directed poly(Phe) synthesis test. The use of homologous tRNAPhe, omission of commercial creatine kinase, and addition of 3.5 mM spermidine at near physiological magnesium concentration (2.5 mM), were the most significant improvements. Under optimal conditions, poly(Phe) synthesis proceeded at a maximal initial rate of 1.2 Phe/80S/min at 37 degrees C, while natural mRNA translation by S-30 started at a near in vivo estimated rate of 0.3-0.5 amino acid/80S/sec. Furthermore, natural mRNA directed the synthesis of a family of polypeptides closely resembling the pattern of cytoplasmic proteins in both, molecular weight and relative amounts. This efficient and faithful system is of interest for biochemical studies of the human translational machinery, as well as a basis for screening new drugs affecting protein synthesis in pathogenic microorganisms.

Chemical Incorporation of 1‐Methyladenosine, Minor tRNA Component, into Oligonucleotides

AbstractFor Abstract see ChemInform Abstract in Full Text.

Synthesis and Conformational Properties of O-β-D-Ribofuranosyl-(1″-2′)-guanosine and (Adenosine)-5″-phosphate

The efficient synthesis of Grp and Arp, minor tRNA components, has been developed.

Chemical Incorporation of 1-Methyladenosine, Minor tRNA Component, into Oligonucleotides

The synthesis of suitably protected 1-methyladenosine derivatives has been developed and its successful chemical incorporation into oligonucleotides was achieved.

Conformationally constrained tricyclic nucleosides. Role of electronic and steric factors

Wyosine [4,9-dihydro-4,6-dimethyl-9-oxo-3-(beta-D-ribofuranosyl) imidazo [1,2-a] purine], a component of tRNA, is known to be constrained in a solution in anti conformation around the glycosylic bond. Guanosine, when substituted with a bulky electronegative group at C8, position analogous to C2 of wyosine, assumes syn conformation. The conformation arrangements of wyosine 2',3',5'-triacetate 1 and its analogs C2 substituted with electronegative groups methoxy, benzyloxy and oxo were studied by diverse 1H NMR techniques. For C2 substituted congeners, significant increase in the North conformer population of sugar moiety was found. It was accompanied by an increase of the tg rotamer contribution at the expense of the gg one. Only in 3 the preferred segment of the anti region was changed and oriented towards high-syn.

Synthesis and Properties of O‐β‐D‐ribofuranosyl‐(1″→2′)‐guanosine‐5″‐ O‐phosphate and Its Derivatives

AbstractThe efficient synthesis of O‐β‐D‐ribofuranosyl‐(1″→2′)‐guanosine‐5″‐O‐phosphate and O‐β‐D‐ribofuranosyl‐(1″→2′)‐adenosine‐5″‐O‐phosphate, minor tRNA components, have been developed, and their conformational properties were examined by NMR spectroscopy.

NMR Studies of Bacillus subtilis tRNATrpHyperexpressed in Escherichia coli: ASSIGNMENT OF IMINO PROTON SIGNALS AND DETERMINATION OF THERMAL STABILITY

15N-Labeled Bacillus subtilis tRNA(Trp) wild type and a series of mutants were hyperexpressed in Escherichia coli and purified for NMR studies with the use of two-dimensional nuclear Overhauser effect spectroscopy (NOESY) and heteronuclear single quantum correlation (HSQC) and three-dimensional NOESY-HSQC techniques. These made possible chemical shift assignments of imino protons and determination of the thermal stability of the tRNA(Trp) molecules. Almost all of the imino protons in the helical regions and the tertiary base pairs were assigned, except three imino protons of the AU base pairs whose peaks were not clearly observed. Several base triplets found in the crystal structure of tRNA were observed in the present study as well. These studies also revealed two components of tRNA(Trp), which could not be separated by high pressure liquid chromatography, corresponding to s(4)U and U at position 8 of the tRNA(Trp), as indicated by two different sets of peaks for the TpsiC and D arms. The modification at position 8 altered the local conformation of the core region of the tRNA. Thermal unfolding experiments showed that the unfolding process is cooperative in the presence of a high concentration of magnesium ions and that the component corresponding to the s(4)U8 is more stable than the U8 component, thus providing evidence that the thiolation of U8 stabilizes the tertiary structure of tRNA.

An Efficient Synthesis and Physico-Chemical Properties OF 2′-O-d-Ribofuranosylnuleosides, Minor tRNA Components

ABSTRACT A high yield preparation of 9-(2-O-β-D-ribofuranosyl-β-D-ribofuranosyl)adenine, guanine- and the pyrimidine analogs (cytosine, thymine and uracil base moiety) has been achieved, and the conformational properties of the ring systems were investigated using NMR spectroscopy and X-ray.

Monoclonal Antibody to Pseudouridine Used to Develop a Radioimmunoassay

Pseudouridine, a component of tRNA, was modified to yield the derivatives: succinyl, palmitoyl pseudouridine, and protein conjugates. These derivatives were used in preparation of monoclonal antibodies specifically directed to pseudouridine. MAbs from three hybridomas (OAL 881, 812 and 814) were established and shown to be directed to pseudouridine, uridine and uracil. OAL 881 was equally reactive with the three substrates, OAL 812 showed the same reactivity for pseudouridine and uracil, while OAL 814 showed a reactivity mainly for pseudouridine. MAb OAL 814 was used in a radioimmunoassay (RIA) system unique for pseudouridine. A good dose-response curve was observed in the range between 31.3 and 2000 nmol/ml. Intra- and inter-assay CV values were below 4.1% and 7.4% respectively, and good results were obtained from recovery of added material and dilution tests. The ratio of pseudouridine/creatinin in urine was significantly higher in patients with cancer than in normal subjects.

Effect of maternal dietary deficiency on the metabolism of high-molecular weight, soluble and transfer RNA in the liver of the 21-day-old offspring of rats

The 21-day-old male progeny of dietary-restricted rats were administered 14C-orotic acid intraperitoneally to study the metabolism of high-molecular weight soluble (sRNA) and transfer RNA (tRNA) in their livers, which were removed at various time intervals ranging from 6 to 120 minutes after the injection. DNA and total RNA were extracted from each liver sample, and nucleic acids were fractionated with 2M NaCl into insoluble (high-molecular weight) and soluble (DNA and tRNA) fractions. All the preparations were analyzed by sucrose density gradient sedimentation, and the distribution of radioactivity was determined. Dietary-restricted progeny experienced significant increases of RNA (p<0.001), including high-molecular weight sRNA (p<0.001) and tRNA components (p<0.001). The greatest increases were apparent in the tRNA and 4–18s RNA fractions. Dietary restriction during the period of growth, gestation and lactation caused a significant reduction in total body weight and liver weight in the 21-day-old progeny. It also enhanced the rate of RNA synthesis in the livers of these animals. The ratios of DNA/total RNA were not affected by dietary deprivation of the progeny, inferring and confirming the observation noted above that the progeny must have experienced augmented RNA degradation and elevated biosynthesis of various forms of RNA.