Selective and sensitive fluorescence "turn-on" detection of 4-thiouridine in nucleic acids via oxidative amination.

A fluorescence "turn-on" method for digestion-free analysis of 4-thiouridine (s4U) in nucleic acids was developed in this work based on the oxidative amination of s4U by fluoresceinamine (FAM-NH2) and periodate (IO4-). It was 125-fold more sensitive for s4U detection than the traditional UV330 absorption method, and showed excellent selectivity to s4U over 2-thiouridine (s2U) analogues and biological thiols.

Monitoring the 5-methoxycarbonylmethyl-2-thiouridine (mcm5s2U) modification in eukaryotic tRNAs via the γ-toxin endonuclease.

The post-transcriptional modification of tRNA at the wobble position is a universal process occurring in all domains of life. In eukaryotes, the wobble uridine of particular tRNAs is transformed to the 5-methoxycarbonylmethyl-2-thiouridine (mcm5s2U) modification which is critical for proper mRNA decoding and protein translation. However, current methods to detect mcm5s2U are technically challenging and/or require specialized instrumental expertise. Here, we show that γ-toxin endonuclease from the yeast Kluyveromyces lactis can be used as a probe for assaying mcm5s2U status in the tRNA of diverse eukaryotic organisms ranging from protozoans to mammalian cells. The assay couples the mcm5s2U-dependent cleavage of tRNA by γ-toxin with standard molecular biology techniques such as northern blot analysis or quantitative PCR to monitor mcm5s2U levels in multiple tRNA isoacceptors. The results gained from the γ-toxin assay reveals the evolutionary conservation of the mcm5s2U modification across eukaryotic species. Moreover, we have used the γ-toxin assay to verify uncharacterized eukaryotic Trm9 and Trm112 homologs that catalyze the formation of mcm5s2U. These findings demonstrate the use of γ-toxin as a detection method to monitor mcm5s2U status in diverse eukaryotic cell types for cellular, genetic, and biochemical studies.

Measuring Transcription Rate Changes via Time-Course 4-Thiouridine Pulse-Labelling Improves Transcriptional Target Identification.

Identifying changes in the transcriptional regulation of target genes from high-throughput studies is important for unravelling molecular mechanisms controlled by a given perturbation. When measuring global transcript levels only, the effect of the perturbation [e.g., transcription factor (TF) overexpression or drug treatment] on its target genes is often obscured by delayed feedback and secondary effects until the changes are fully propagated. As a proof of principle, we show that selective measuring of transcripts that are only synthesised after a perturbation [4-thiouridine (4sU) sequencing (4sU-seq)] is a more sensitive method to identify targets and time-dependent transcriptional responses than global transcript profiling. By metabolically labelling RNA in a time-course setup, we could vastly increase the sensitivity of MYCN target gene detection compared to traditional RNA sequencing. The validity of targets identified by 4sU-seq was demonstrated using chromatin immunoprecipitation sequencing and neuroblastoma microarray tumour data. Here, we describe the methodology, both molecular biology and computational aspects, required to successfully apply this 4sU-seq approach.

Familial dysautonomia (FD) patients have reduced levels of the modified wobble nucleoside mcm(5)s(2)U in tRNA.

Familial dysautonomia (FD) is a recessive neurodegenerative genetic disease. FD is caused by a mutation in the IKBKAP gene resulting in a splicing defect and reduced levels of full length IKAP protein. IKAP homologues can be found in all eukaryotes and are part of a conserved six subunit protein complex, Elongator complex. Inactivation of any Elongator subunit gene in multicellular organisms cause a wide range of phenotypes, suggesting that Elongator has a pivotal role in several cellular processes. In yeast, there is convincing evidence that the main role of Elongator complex is in formation of modified wobble uridine nucleosides in tRNA and that their absence will influence translational efficiency. To date, no study has explored the possibility that FD patients display defects in formation of modified wobble uridine nucleosides as a consequence of reduced IKAP levels. In this study, we show that brain tissue and fibroblast cell lines from FD patients have reduced levels of the wobble uridine nucleoside 5-methoxycarbonylmethyl-2-thiouridine (mcm(5)s(2)U). Our findings indicate that FD could be caused by inefficient translation due to lower levels of wobble uridine nucleosides.

Genome-wide technology for determining RNA stability in mammalian cells: historical perspective and recent advantages based on modified nucleotide labeling.

Changing the abundance of transcripts by regulated RNA degradation is a critical step in the control of various biological pathways. Recently, genome-wide inhibitor-free technologies for determining RNA stabilities in mammalian cells have been developed. In these methods, endogenous RNAs are pulse labeled by uridine analogs [e.g., 4-thiouridine (4sU), 5-etyniluridine (EU) and 5'-bromo-uridine (BrU)], followed by purification of labeled de novo RNAs. These technologies have revealed that the specific half-life of each mRNA is closely related to its physiological function. Genes with short-lived mRNAs are significantly enriched among regulatory genes, while genes with long-lived mRNAs are enriched among housekeeping genes. This review describes the recent progress of experimental procedures for measuring RNA stability.

Transient reduction in the tRNA 4-thiouridine content induced by ultraviolet A during post-irradiation growth in Enterobacter cloacae.

The influence of previous exposure to ultraviolet-A radiation (UVA) was studied on the susceptibility of Enterobacter cloacae to undergo the growth delay effect. Comparison of growth curves corresponding to irradiated and control cells showed that a previous treatment with UVA almost abolished the growth delay effect. UV absorption spectra of tRNA, and reverse phase HPLC analysis of hydrolysed tRNA, demonstrated a low content of 4-thiouridine in E. cloacae cells grown after UVA exposure at low doses. Since 4-thiouridine is the UVA target responsible for initiation of growth delay, this observation explained the influence of previous exposure to UVA on the susceptibility of this organism to undergo growth delay. A similar but weaker alteration was found when Escherichia coli was assayed. The results suggest that, in addition to cross-linking with cytidine residues, the content of 4-thiouridine in tRNA may be modified by UVA by an unknown mechanism.

Wobble modification defect in tRNA disturbs codon-anticodon interaction in a mitochondrial disease.

We previously showed that in mitochondrial tRNA(Lys) with an A8344G mutation responsible for myoclonus epilepsy associated with ragged-red fibers (MERRF), a subgroup of mitochondrial encephalomyopathic diseases, the normally modified wobble base (a 2-thiouridine derivative) remains unmodified. Since wobble base modifications are essential for translational efficiency and accuracy, we used mitochondrial components to estimate the translational activity in vitro of purified tRNA(Lys) carrying the mutation and found no mistranslation of non-cognate codons by the mutant tRNA, but almost complete loss of translational activity for cognate codons. This defective translation was not explained by a decline in aminoacylation or lowered affinity toward elongation factor Tu. However, when direct interaction of the codon with the mutant tRNA(Lys) defective anticodon was examined by ribosomal binding analysis, the wild-type but not the mutant tRNA(Lys) bound to an mRNA- ribosome complex. We therefore concluded that the anticodon base modification defect, which is forced by the pathogenic point mutation, disturbs codon- anticodon pairing in the mutant tRNA(Lys), leading to a severe reduction in mitochondrial translation that eventually could result in the onset of MERRF.

Tertiary structural analysis of Escherichia coli lysine tRNA.

It has been suggested that Escherichia coli lysine tRNA (E. coli tRNA(Lys)) has an unusual anticodon loop structure, judging from the abnormal CD pattern derived from 5-methylaminomethyl-2-thiouridine (mnm5s2U) located at the first position of the anticodon of the tRNA. Here, the structure of E. coli tRNA(Lys) was analysed by chemical modification and nuclear magnetic resonance (NMR) of the imino protons. The chemical probing approach showed that the anticodon arm region forms the ordinary stem-and-loop structure as expected from the standard clover-leaf structure, because N7 of G30, N3 of C40, N7 of A41 and N7 of A42 were protected under a native condition. By using the heteronuclear 15N-1H single quantum coherence (HSQC) spectra of 15N-labeled E.coli tRNA(Lys) and the analyses of the nuclear Overhauser effects (NOE) between the imino protons of non labeled E.coli tRNA(Lys), the imino protons in the acceptor stem was assigned, which indicates that the acceptor stem exists in an usual form. The tertiary base-base interactions was also detected. Thus, E.coli tRNA(Lys) seems to maintain a similar structure to the ordinary L-shaped tertiary structure, as a whole. The fine structure of the anticodon loop is now being analyzed.

Selenium-containing tRNA(Glu) and tRNA(Lys) from Escherichia coli: purification, codon specificity and translational activity.

In response to low (approximately 1 microM) levels of selenium, Escherichia coli synthesizes tRNA(Glu) and tRNA(Lys) species that contain 5-methylaminomethyl-2-selenouridine (mnm5Se2U) instead of 5-methylaminomethyl-2-thiouridine (mnm5S2U). Purified glutamate- and lysine-accepting tRNAs containing either mnm5Se2U (tRNA(SeGlu), tRNA(SeLys] or mnm5S2U (tRNA(SGlu), tRNA(SLys] were prepared by RPC-5 reversed-phase chromatography, affinity chromatography using anti-AMP antibodies and DEAE-5PW ion-exchange HPLC. Since mnm5Se2U, like mnm5S2U, appears to occupy the wobble position of the anticodon, the recognition of glutamate codons (GAA and GAG) and lysine codons (AAA and AAG) was studied. While tRNA(SGlu) greatly preferred GAA over GAG, tRNA(SeGlu) showed less preference. Similarly, tRNA(SGlu) preferred AAA over AAG, while tRNA(SeLys) did not. In a wheat germ extract--rabbit globin mRNA translation system, incorporation of lysine and glutamate into protein was generally greater when added as aminoacylated tRNA(Se) than as aminoacylated tRNA(S). In globin mRNA the glutamate and lysine codons GAG and AAG are more numerous than GAA and AAA, thus a more efficient translation of globin message with tRNA(Se) might be expected because of facilitated recognition of codons ending in G.

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.

5-Methyl-2-thiouridine in the tRNA of Candida tropicalis and its localization in lysine tRNA.

35S incorporation studies showed that Candida tropicalis tRNA contained two thionucleosides, one of which was identified as 5-methyl-2-thiouridine. The other thionucleoside was alkali labile, and it appeared to be an ester. Pulse-chase experiments suggested that the two thionucleosides were structurally related. 5-Methyl-2-thiouridine was present in one of the lysine tRNAs. This is the first report of the presence of this nucleoside in a yeast tRNA.

Chemical conversion of cytidine residues into 4-thiouridines in yeast tRNAPhe. Determination of the modified cytidines.

Treatment of yeast phenylalanine tRNA with pressurized hydrogen sulfide results in conversion of cytidine residues into 4-thiouridine residues. Under conditions leading to an average modification of one cytidine per tRNA molecule 9 positions are thiolated. The 4-thiouridine residues are distributed along the tRNA molecule. Four of the reactive cytidines are located in single-stranded regions: Cm32 , C60 , C74 and C75 . The five others are located in base pairs: C2, C27, C56 , C61 and C63 . Importance of replacement of an amino group by a thiol group on hydrogen bonding and on biological activity of the modified tRNA is discussed.

Thiolated nucleotides in yeast transfer RNA.

By culturing Saccharomyces cerevisiae in growth medium containing Mg35SO4, we have determined the extent and variation of tRNA thiolation in this yeast. We find that 5-methoxycarbonylmethyl-2-thiouridine (mcm5s2U)1 is the major, if not only, thiolated derivative in S. cerevisiae tRNA. In addition, a comparison of the chromatographic mobility of mcm5s2Up on cellulose thin layers with those reported for unknown uridine derivatives found in purified yeast tRNA digests, leads to the conclusion that at least two of these tRNAs contain this modification.

Separation and comparison of 2-thioribothymidine-containing transfer ribonucleic acid and the ribothymidine-containing counterpart from cells of Thermus thermophilus HB 8.

For the extreme thermophile Thermus thermophilus HB 8, a positive correlation was observed among the growth temperatures of the cells, the melting temperature, and the 2-thioribothymidine (s2T) content of tRNA extracted from cells grown at various temperatures [Watanabe, K., Shinma, M., Oshima, T., & Nishimura, S. (1976) Biochem. Biophys. Res. Commun. 72, 1137-1144]. On the basis of these observations, studies were carried out from which the following results were obtained. (1) Both RNase T1 and U2 digestions of tRNA gave only four fragments containing s2T or T: s2T psi CGp, s2T psi CAp, T psi CGp, and T psi CAp. For the different growth temperatures, the ratio of the content of s2T psi CGp plus s2T psi CAp to that of T psi CGp plus T psi CAp was almost the same as that of the s2Tp to Tp content reported previously. (2) The midpoint of the s2T-specific circular dichroism spectral change induced by heat was constant for all tRNAs extracted from cells grown at various temperatures, suggesting that the s2T-containing tRNAs melt at about the same temperature, which is independent of the growth temperature of cells. (3) s2T-containing tRNA was separated from the T-containing counterpart quantitatively by a specific modification of s2T with bromoaceto-2,4-dinitroanilide followed by BD-cellulose column chromatography. The molar ratio of the s2T- and T-containing tRNAs was also similar to that of s2Tp to Tp as mentioned above. These results demonstrate that T. thermophilus cells have both s2T- and T-containing tRNAs, whose relative content is controlled by the growth temperature. This phenomenon may be necessary to enable the thermophile to adapt to higher temperatures.

Thiolation and 2-methylthio- modification of Bacillus subtilis transfer ribonucleic acids.

Six thionucleosides found in Bacillus subtilis transfer ribonucleic acids were investigated: N6-(delta 2-isopentenyl)-2-methylthioadenosine, 5-carboxymethylaminomethyl-2-thiouridine, 4-thiouridine, 2-methylthioadenosine, N-[(9-beta-D-ribofuranosyl-2-methylthiopurin-6-yl)carbamoyl]threonine, and one unknown (X1). The presence of N-[(9-beta-D-ribofuranosyl-2-methylthiopurin-6-yl)carbamoyl]threonine was demonstrated based on the affinity of the transfer ribonucleic acid containing it for an immunoadsorbent made with the antibody directed toward N-[9-(beta-D-ribofuranosyl)purin-6-ylcarbamoyl]-L-threonine. The existance of N-[(9-beta-D-ribofuranosyl-2-methylthiopurin-6-yl)carbamoyl]threonine in two species of lysine transfer ribonucleic acids was also confirmed by high-resolution mass spectrometry. Four of these thionucleosides--N6-(delta 2-isopenenyl)-2-methylthioadenosine, 2-methylthioadenosine, 5-carboxymethylaminomethyl-2-thiouridine, and the unknown designated X1--occurred only in specific areas in the elution profile of an RPC-5 column and probably affect the chromatographic properties of the transfer ribonucleic acids containing them. In contrast with Escherichia coli, where 4-thiouridine is the most frequent type of sulfur-containing modification, approximately one-third of the sulfur groups in B. subtilis transfer ribonucleic acid are present as thiomethyl groups on the 2 position of an adenosine or modified adenosine residue.

tRNA thiolated pyrimidines as targets for near-ultraviolet-induced synthesis of guanosine tetraphosphate in Escherichia coli.

Illumination with near-ultraviolet light triggers synthesis of ppGpp (guanosine 3'-diphosphate 5'-diphosphate) not only in growing Escherichia coli cells containing the putative chromophore 4-thiouridine in their tRNAs [Ramabhadran, T. V and Jagger, J. (1976) Proc. Natl Acad. Sci. USA, 73, 59--69], but also in nuv- cells which lack 4-thiouridine. The burst of ppGpp in nuv- cells is, however, induced exclusively by light of wavelengths shorter than 350 nm. Its maximum level is half that obtained in the parental strain. This ppGpp synthesis is also under the control of the relA gene, indicating that it is due to the accumulation of uncharged tRNAs. A candidate likely to trigger this effect is a 5-methylaminomethyl-2-thiouracil residues present in the first position of the anticodon loop of tRNAGlu, tRNALys and one tRNAGln isoacceptor. In conditions in vitro, this base is highly photoreactive at wavelengths shorter than 350 nm. Furthermore, near-ultraviolet-photomodified tRNAGlu and tRNALys become poor substrates of their acylation enzyme.

Identification of modified nucleosides in intact transfer ribonucleic acid by pyrolysis-electron impact-collisional activation mass spectrometry.

A novel mass spectrometric method has been developed for the detection and identification of dihydrouridine, ribothymidine, 4-thiouridine, and 7-methylguanosine in Escherichia coli tRNAs. The method utilizes (a) Pyrolysis-Electron Impact-Mass Spectrometry (PYEIMS), a procedure which releases the purine and pyrimidine bases from the intact, underivatized tRNA molecule. The mass spectrum exhibits intense peaks for the bases deriving from the common nucleosides in tRNA as well as peaks of much lower intensity at mass values expected for the bases from modified components known to be present in the tRNA; and, (b) Collisional Activation Mass Spectrometry (CAMS), a technique which permits the isolation of a single ion species from a complex mass spectrum. Subsequent fragmentation of that species yields a characteristic collisional activation spectrum. Such analyses of the ion species that were presumed to originate from H2Urd, rThd, 4SUrd, and 7MeGuo in the tRNA were used to define the structure and, thus, the identity of each component. Attributes of the PYEICAMS technique are that (a) precise structural elucidation of minor nucleosides present in tRNAs at the 1 - 4% level is obtained; (b) the high order of sensitivity allows the analysis to be done on microgram amounts of tRNA; and (c) there is no requirement for enzymatic or chemical hydrolysis of the tRNA or for subsequent chromatographic separation methods.

5-(carboxymethylaminomethyl)-2-thiouridine, a new modified nucleoside found at the first letter position of the anticodon.

The structure of a modified uridine derivative which was detected at the first letter position of the anticodon of Bacillus subtilis tRNA1Lys was determined to be 5-(carboxymethylaminomethyl)-2-thiouridine. The determination was mainly based in this ultraviolet absorption spectra and mass spectrometric analysis of the trimethylsilyl derivative.

Formation of the [b+41]+. ion in the mass spectra of 2-thiouridines.

In the electron impact mass spectra of some 2-thiouridines a prominent base +41 (b+C2HO) peak was found. The sulphur atom at the 2 position facilitates its formation.