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At least 19 recordsLinked to original sources
PubMed1977

Pseudouridine-deficient transfer RNAs from Escherichia coli B and their use as substrates for pseudouridine synthetase.

Transfer RNAs isolated from Escherichia coli B grown in the presence of 2-thiouracil are deficient in pseudouridine. Much of this deficiency is from the T psi C region, which has only about 50% of its normal pseudouridine content. The other modified nucleoside from this region, ribothymidine, is reduced by only about 10%. Studies showed that 2-thiouracil is incoproated into the RNA of E. coli during growth in the presence of the analog. This incorporation appears to result from the replacement of uracil, occur in a random manner, and involve all RNA species. The extent of incorporation varies from 1 to 3 mol %, depending upon the preparation and RNA species examined. Electrophoresis on polyacrylamide gels and chromatography on Sephadex G-75 and reverse phase (Systen 5) columns of normal and 2-thiouracil-containing tRNAs revealed no profile differences. No accumulation of any precursor tRNA in the thiopyrimidine-treated cells is found. A partial recovery of the pseudouridine content of 2-thiouracil-containing tRNAs can be achieved in vivo by removal of the 2-thiouracil from the culture media. These transfer RNAs have also been used as substrates to study the properties of a partially purified preparation of pseudouridine synthetase II invitro and should be useful as substrates in the further purification of this enzyme.

Escherichia coli↗
PubMed1978

The pseudouridine contents of the ribosomal ribonucleic acids of three vertebrate species. Numerical correspondence between pseudouridine residues and 2'-O-methyl groups is not always conserved.

The pseudouridine contents of the rRNA species of HeLa cells, mouse L-cells and Xenopus laevis cultured kidney cells were examined. Pseudouridine, like 2'-O-methylation, was found to occur relatively frequently in each of the high-molecular-weight rRNA species. However, the numerical data do not support the idea that there is a general one-to-one relationship between pseudoridine residues and 2'-O-methyl groups in vertebrate rRNA.

Animals↗
PubMedDate not supplied

[Interrelationship of pseudouridine and transfer RNA metabolic disorders in thermal injury].

Excretion and turnover rate of radioactive pseudouridine were studied in intact and burned rats. The thermic injury increased the pseudouridine excretion and lowered the rate of decrease in its specific radioactivity. Relative increase in excretion of pseudouridine was more distinct for the "B" isomer as compared with the isomer "C". The general pattern of dynamics of alterations in pseudouridine turnover after thermic injury corresponded to the patterns of impairments in metabolism of transfer RNA, which were studied previously. But the rate of pseudouridine turnover considerably exceeded the rate of tRNA metabolism either in normal state or in thermic injury.

Animals↗
PubMed2026

The pseudouridine epitranscriptomic landscape of advanced prostate cancer therapeutic resistance identifies TIMM17A as a key player.

BACKGROUND: Resistance to androgen receptor signaling inhibitors (ARSIs) remains a major barrier of advanced prostate cancer (PCa) treatment. While RNA epitranscriptomic modifications are increasingly recognized as key regulators of tumor biology, the role of pseudouridine (Ψ) in therapeutic resistance is largely unexplored. METHODS: A darolutamide-resistant PCa cell model was established and subjected to integrated multi-omics profiling using bulk RNA sequencing and photo-crosslinking-assisted Ψ sequencing (PA-Ψ-seq). Differential expression and pseudouridylation analyses were combined to identify Ψ-associated genes. Public datasets validated expression and prognosis. Functional assays including RNA knockdown, cell proliferation, colony formation, and xenograft models were conducted. Single-cell RNA sequencing investigated tumor microenvironment (TME) interactions. RESULTS: We identified extensive transcriptomic and pseudouridylation alterations associated with ARSI resistance, with a significant positive correlation between Ψ modification and mRNA expression. Integrated analysis highlighted a subset of "hyper-up" genes enriched in resistance-related pathways. Thus, TIMM17A was identified as a novel candidate. TIMM17A expression was significantly elevated in PCa and correlated with disease progression and poor prognosis. Experimental validations demonstrated that TIMM17A promoted tumor growth and resistance, while its knockdown restored sensitivity to darolutamide both in vitro and in vivo. Mechanistically, TIMM17A expression may be regulated by PUS1‑mediated pseudouridylation. Single-cell analysis further revealed that TIMM17A is enriched in malignant epithelial cells and associated with enhanced cell-cell communication within the TME. CONCLUSIONS: This study delineates the pseudouridine epitranscriptomic landscape in advanced PCa and identifies TIMM17A as a key mediator of therapeutic resistance. Targeting the Ψ-TIMM17A axis may offer a novel strategy to overcome ARSI resistance.

Advanced prostate cancer↗
PubMed1978

Replacement of pseudouridine in transfer RNA by 5-fluorouridine does not affect the ability to stimulate the synthesis of guanosine 5'-triphosphate 3'-diphosphate.

The requirement for ribothymidine and pseudouridine in the TpsiCG loop of tRNA for its activity in the ribosome and tRNA-stimulated synthesis of guanosine 5'-triphosphate 3'-diphosphate (pppGpp) by stringent factor has been tested by the use of a purified tRNAPhe (883 pmol of phenylalanine incorporated/A260 unit) in which 92% of the pseudouridine, 98% of the ribothymidine, 98% of the dihydrouridine, and 88% of the uridines were substituted by 5-fluorouridine. This tRNA was quantitatively as active as control tRNA in inducing pppGpp synthesis. With loose-couple ribosomes, the concentration of tRNA needed to give half-maximal reaction was 0.07 micrometer for both normal and fluorouridine-substituted tRNA, with vacant tight-couple ribosomes it was 0.05 micrometer, and with tight couples carrying poly(Phe)-tRNA at the P site the value was 0.02 micrometer. These results show that at the level of intact tRNA there is no special requirement for modified bases in the TpsiCG loop of tRNA in the synthesis of pppGpp.

Escherichia coli↗
PubMed1979

Pyrimidine nucleoside, pseudouridine, and modified nucleoside excretion by growing and resting fibroblasts.

We are examining the relationship of RNA metabolism and de novo pyrimidine synthesis as parameters of malignant transformation. These initial experiments on normal hamster embryo fibroblasts have shown that excreted nucleosides are markers for intracellular RNA metabolism. We employed affinity chromatography to concentrate the nucleosides in the medium and sensitive column chromatographic procedures to quantitatively measure them. The excretion of pyrimidine nucleoside from hamster embryo fibroblasts in sulture was found to be dependent on the growth state of the cells, with the greatest accumulation occurring cell quiescence. The major nucleoside excretion products, uridine and cytidine, were both normal end products of RNA metabolism and the major nucleoside excretion products from cultured cells. The modified nucleosides N-1-methylguanosine, N-2-methylguanosine, N-2-dimethylguanosine, N-4-acetylcytidine, N-1-methylinosine, pseudouridine, N-1-methyladenosine, N-3-methylcytidine, and 5-methyleycytidine were found, as were several unidentified nucleosides.

Animals↗
PubMed2026

Meta-PseU: A meta-classifier for robust prediction of RNA pseudouridine modification sites from long sequences.

BACKGROUND AND OBJECTIVES: Pseudouridine (Ψ) represents one of the most abundant and conserved RNA modifications. Ψ provides an additional hydrogen-bond donor that enhances RNA structural stability and modulates translation. It participates in diverse biological processes, including RNA-protein interactions, splicing, translational control, and stress responses. Aberrant pseudouridylation is implicated in cancer, neurodegenerative disorders, and autoimmune diseases. Despite its biological importance, experimental identification of Ψ sites remains time-consuming and costly, limiting the feasibility of transcriptome-wide profiling. Computational approaches have therefore become essential complements to experimental techniques. However, state-of-the-art machine-learning and deep-learning predictors often suffer from limited generalizability due to small training datasets. To overcome these issues, we aim at constructing new long-sequence datasets and developing a novel Ψ site predictor. METHODS: New long-sequence datasets were constructed as benchmarks for RNA Ψ-site prediction. The Ψ modification sites in RMBase 3.0 were mapped to the reference genomes across three species of human, mouse, and yeast, and the RNA sequences with a length of 201 were generated by extending the upstream and downstream from the mapped, central sites. To eliminate sequence redundancy, the sequences were clustered using CD-HIT with a 70% sequence identity threshold. We developed Meta-PseU, a logistic regression-based meta-classifier that considered 118 machine learning and deep learning classifiers. The datasets and programs are freely accessible at https://github.com/kuratahiroyuki/MetaPseU. RESULTS: By optimizing model configuration, we proposed the Meta-PseU model stacking 32 machine learning and deep learning classifiers out of 118 classifiers. Meta-PseU substantially improved model generalizability, overcoming a key limitation of existing approaches. It greatly outperformed state-of-the-art predictors and achieved increasing accuracy with increasing sequence length. CONCLUSIONS: Long-sequence datasets were newly constructed as benchmarks for RNA Ψ-site prediction. Meta-PseU offers a new framework for robust Ψ-site identification by using long sequences.

Pseudouridine↗
PubMed1976

A heterologous system for detecting eukaryotic enzymes which synthesize pseudouridine in transfer ribonucleic acids.

tRNA pseudouridylation activities have been detected in embryonic mouse cell fractions and in extracts from HeLa, mouse L-cell and baby hamster kidney (BHK) cell lines. These activities were identified by the use of heterologous reaction systems, with tRNA from hisT strains of Salmonella typhimurium as substrate. hisT mutants are defective for an enzyme that forms psi residues in the anticodon region of many tRNAs and accumulate undermodified species of tRNA. The pseudouridylation activity from BHK cells has been examined in detail and quantitated by a modified tritium release assay (Cortese, R., Kammen, H.O., Spengler, S.J., and Ames, B.N. (1974) J. Biol. Chem. 249, 1103-1108). Maximal rates of tritium release required a suitable cationic environment (optimally, a combination of Mg2+ and NH4+) and a thiol reductant. The activity was totally inhibited in the presence of thiol-reactive reagents, such as 5,5'-dithiobis(2-nitrobenzoic acid) and p-chloromercuribenzoate. A major portion of this 3H release activity was associated with psi modification reactions. This conclusion stems from the following observations: (a) BHK extracts preferentially catalyzed a release of 3H from hisT [5-3H]tRNA, rather than from similarly labeled wild type tRNA; (b) this activity was specific for protons attached to C5 of the pyrimidine rings; no release of 3H was obtained with hisT or wild type [6-3H]tRNA as substrate; (c) the reaction products of hisT tRNA with BHK enzyme were examined by reverse phase column chromatography of tRNAPhe isoacceptors on RPC-5 columns. The enzyme modified both of the principal isoacceptors of hisT tRNAPhe to an equal extent, yielding products indistinguishable from wild type tRNAPhe. Significant levels of 3H release were obtained by the action of enzyme on wild type [5-3H]tRNA, even after gel filtration of the enzyme. This suggests that the enzyme may be able to hypermodify certain species of wild type S. typhimurium tRNA. The activities for wild type tRNA and hisT tRNA appeared to be associated with the same enzyme.

Base Sequence↗
PubMed1977

Modification-deficient transfer ribonucleic acids from relaxed control Escherichia coli: structures of the major undermodified phenylalanine and leucine transfer RNAs produced during leucine starvation.

The structures of the major, chromatographically unique phenylalanine and leucine tRNAs produced during leucine starvation of a relaxed control (rel-) mutant of E. coli have been determined. The results demonstrate that the unique species are modification-deficient forms of the major, normally occurring isoacceptor species. The unique tRNAphe differs from the fully modified species at nucleotide positions 16, 37, 39, 47, and 55 from the 5' terminus. The unique species contains uridine (U) in place of dihydrouridine-16 (D16), isopentenyladenosine in place of 2-thiomethyl-N6-(delta2-isopentenyl)adenosine-37, a mixture of U and pseudouridine (psi) in position 39, a mixture of U and 3-(3-carboxypropyl)uridine at position 47, and a mixture of U and psi at position 55. The chromatographically normal isoacceptor from amino acid starved cells is deficient in D16 and psi55, indicating that that species is a mixture of mature and undermodified tRNAs. The unique tRNALeu isoacceptor consists of two subspecies which are undermodified forms of the major, normally occurring isoacceptor, tRNALeuI. Both unique subspecies lack the D and psi residues which occur at positions 16 and 39 from the 5' terminus; one subspecies also lacks D17. Compared with the tRNALeusI from wild-type strains of E. coli B and K12, both tRNALeuI from nonstarved cells and the unique, rel-tRNALeu are deficient in the modified guanosine which normally occurs adjacent to the anticodon and the pseudouridine in the GTpsiC sequence of the psi loop. Both the unique tRNAPhe and the unique tRNALeu lack dihydrouridine residues which occur in the 5' half of the D loop and pseudouridines which occur in the 3' half of the anticodon loop and adjoining stem. Taken together, these findings suggest that the same enzymes are responsible for the formation of these particular modified bases in both tRNAs. The results further suggest that several, perhaps most, of the tRNAs from cells cultured under conditions in which RNA and protein synthesis are uncoupled will be similarly deficient in dihydrouridine and pseudouridine and other minor nucleosides which occur less frequently. Because both modification-deficient rel-tRNAs have dihydrouridine at position 20 and pseudouridine in the psi loop (and at position 41 in the unique tRNALeu), the results support the view that there was multiple D-and psi-forming enzymes in E. coli, some of which may turn over rapidly or are selectively inactivated when protein synthesis is blocked. The results are discussed with a view toward understanding the structural basis for the altered biological activity of the unique tRNAPhe species and the order of events in the posttranscriptional modification of newly synthesized tRNA.

Base Sequence↗
PubMed1979

Pseudouridylation of yeast ribosomal precursor RNA.

The pseudouridylation of ribosomal RNA of Saccharomyces carlsbergensis was investigated with respect to its timing during the maturation of rRNA and its sequence specificity. Analysis of 37-S RNA, the common precursor to 17-S, 5.8-S and 26-S rRNA and most probably the primary ribosomal transcript, shows that this RNA molecule contains already most if not all of the 36-37 pseudouridine residues found in the mature rRNAs. Thus pseudouridylation is, like 2'-0-ribosemethylation, an early event in the maturation of rRNA, taking place immediately after, or even during, transcription. The data presented show that the non-conserved sequences of 37-S precursor rRNA contain very few pseudouridine residues if any. The pseudouridine residues within the rRNA sequences are apparently clustered to a certain degree as can inferred from the occurrence of a single oligonucleotide containing 3 pseudouridines, which was obtained by digestion of 26-S rRNA with ribonuclease T1.

Base Sequence↗