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PubMed2002

An RNA complex of the HIV-1 A-loop and tRNA(Lys,3) is stabilized by nucleoside modifications.

The HIV transcription initiation complex involves a putative interaction between the primer tRNA anticodon and a conserved A-rich loop in the HIV genome. Surface plasmon resonance was used to demonstrate that the hypermodified nucleosides in the tRNA anticodon stem loop (ASL) stabilize RNA-RNA interactions in a model for the anticodon/A-loop complex. tRNA ASL hairpins with the modifications of Escherchia coli tRNALys and human tRNALys,3 each form stable complexes. Partially modified tRNA ASLs bind the A-loop hairpin with lesser affinity, and it was found that the modifications of the bacterial and mammalian tRNAs make distinct contributions toward stabilizing the RNA complex. One model for the anticodon/A-loop RNA complex that is consistent with the known modification effects on tRNA structure and function is that of complementary tRNAs, as seen for the published crystal structure of tRNAAsp.

Adenosine↗
PubMed1983

Tumor markers derived from nucleic acid components.

It was known for some time that cancer patients excrete in their urine elevated levels of modified nucleosides. From earlier work, we were able to show that most of these modified nucleosides originate from transfer RNA (tRNA). The modifications are achieved at the macromolecular level by enzymes after primary synthesis. Such modifications are highly specific and, therefore, when the modified nucleosides accumulate from the tRNA breakdown, they cannot be reinserted randomly by the polymerases and must be excreted. We found that the modifying enzymes are aberrantly hyperactive in every malignant tissue. We also found that there is abnormally high turnover of tRNA in malignant tissues, which is probably the source of the elevated levels of excretion products. Since these products originate from a cardinal component of the molecular biology of every cell, the determination of markers in the urine may be a universal indicator of malignancy. We are focusing on the use of these markers in syndromes whose diagnoses are otherwise difficult. Since the marker levels return to normal very soon after chemotherapy, such determinations can be used to monitor the effectiveness of therapy. Therefore, the clinical oncologists can adapt their protocols to the specific need of a patient.

Chorionic Gonadotropin↗
PubMed2026

Phosphate backbone epitranscriptomics: Discovery of natural RNA phosphorothioates and their writer machinery.

Over 150 modifications expand the RNA alphabet, yet all known natural modifications occur on nucleobases or ribose sugars, with none identified on the phosphate backbone. In contrast, phosphorothioates (PSs), in which a non-bridging phosphate oxygen is replaced with sulfur, are central to RNA therapeutics but have never been reliably detected in natural RNAs. Here, we develop sequencing- and mass spectrometry-based approaches to quantitatively map RNA PSs at single-nucleotide resolution. Across diverse archaeal species, we identify stereospecific PS modifications at rRNA and tRNA hotspots, which are dynamically regulated by sulfur availability and temperature. We uncover a diverse enzyme family that selectively modifies tRNA/rRNA substrates and whose evolutionary presence/absence matches the distribution of PSs. Enzyme loss causes inviability or temperature sensitivity, and functional analyses reveal that tRNA PSs enhance tRNA stability. These findings establish the first natural RNA phosphate-backbone modification and its enzymatic machinery, providing a foundation for mechanistic and functional exploration.

RNA modifications↗
PubMed1993

Structure of the archaeal transfer RNA nucleoside G*-15 (2-amino-4,7-dihydro- 4-oxo-7-beta-D-ribofuranosyl-1H-pyrrolo[2,3-d]pyrimidine-5-carboximi dam ide (archaeosine)).

A number of post-transcriptional modifications in tRNA are phylogenetically characteristic of the bacterial, eukaryal, or archaeal domains, both with respect to sequence location and molecular structure at the nucleoside level. One of the most distinct such modifications is nucleoside G*, located in archaeal tRNA at position 15, which in bacterial and eukaryal tRNAs is a conserved site involved in maintenance of the dihydrouridine loop-T-loop tertiary interactions. G* occurs widely in nearly every branch of the archaeal phylogenetic domain, in contrast to its absence in all reported bacterial and eukaryal tRNA sequences. The structure of G*-15 is 2-amino-4,7-dihydro-4-oxo-7-beta-D-ribofuranosyl-1H- pyrrolo[2,3-d]pyrimidine-5-carboximidamide (7-formamidino-7-deazaguanosine), which is a non-purine, non-pyrimidine ribonucleoside; its structure thus reflects extensive modification beyond the guanine-15 specified by corresponding gene sequences. The structure was established by mass spectrometry, and in particular from collision-induced dissociation mass spectra of derivatives formed by microscale permethylation, and is confirmed by chemical synthesis.

Chromatography, High Pressure Liquid↗
PubMed1989

Altered queuine modification of transfer RNA involved in the in vitro transformation of Chinese hamster embryo cells.

Altered queuine modification of tRNA has been correlated to neoplastic transformation, but no direct cause and effect relationship has been defined. In the present study, a potential role for this alteration has been assigned. The tRNA in normal Chinese hamster embryo cells is significantly more queuine modified than the tRNA in their transformed Chinese hamster embryo counterparts, even though the specific activity of the queuine modification enzyme is much lower in Chinese hamster embryo cells than in transformed Chinese hamster embryo cells. Substrate availability appears to be responsible for the queuine hypomodification of tRNA in the transformed cells, since addition of excess exogenous queuine to the culture medium results in incorporation of queuine into the anti-codon of the undermodified tRNAs. Most importantly, the excess queuine inhibits anchorage-independent growth of transformed Chinese hamster embryo cells, thereby implicating queuine hypomodification of tRNA in the expression of this transformed phenotype.

Animals↗
PubMed2001

Specific interaction between anticodon nuclease and the tRNA(Lys) wobble base.

The bacterial tRNA(Lys)-specific PrrC-anticodon nuclease cleaves its natural substrate 5' to the wobble base, yielding 2',3'-cyclic phosphate termini. Previous work has implicated the anticodon of tRNA(Lys) as a specificity element and a cluster of amino acid residues at the carboxy-proximal half of PrrC in its recognition. We further examined these assumptions by assaying unmodified and hypomodified derivatives of tRNA(Lys) as substrates of wild-type and mutant alleles of PrrC. The data show, first, that the anticodon sequence and wobble base modifications of tRNA(Lys) play major roles in the interaction with anticodon nuclease. Secondly, a specific contact between the substrate recognition site of PrrC and the tRNA(Lys) wobble base is revealed by PrrC missense mutations that suppress the inhibitory effects of wobble base modification mutations. Thirdly, the data distinguish between the anticodon recognition mechanisms of PrrC and lysyl-tRNA synthetase.

Alleles↗
PubMed2002

EFG-independent translocation of the mRNA:tRNA complex is promoted by modification of the ribosome with thiol-specific reagents.

Translation of polyphenylalanine from a polyuridine template by the ribosome in the absence of the elongation factors EFG and EFTu (and the energy derived from GTP hydrolysis) is promoted by modification of the ribosome with thiol-specific reagents such as para-chloromercuribenzoate (pCMB). Here, we examine the translational cycle of modified ribosomes and show that peptide bond formation and tRNA binding are largely unaffected, whereas translocation of the mRNA:tRNA complex is substantially promoted by pCMB modification. The translocation movements that we observe are authentic by multiple criteria including the processivity of translation, accuracy of movement (three-nucleotide) along a defined mRNA template and sensitivity to antibiotics. Characterization of the modified ribosomes reveals that the protein content of the ribosomes is not depleted but that their subunit association properties are severely compromised. These data suggest that molecular targets (ribosomal proteins) in the interface region of the ribosome are critical barriers that influence the translocation of the mRNA:tRNA complex.

Anti-Bacterial Agents↗
PubMed2004

Yeast Nfs1p is involved in thio-modification of both mitochondrial and cytoplasmic tRNAs.

The IscS protein is a pyridoxal phosphate-containing cysteine desulfurase involved in iron-sulfur cluster biogenesis. In prokaryotes, IscS is also involved in various metabolic functions, including thio-modification of tRNA. By contrast, the eukaryotic ortholog of IscS (Nfs1) has thus far been shown to be functional only in mitochondrial iron-sulfur cluster biogenesis. We demonstrate here that yeast Nfs1p is also required for the post-transcriptional thio-modification of both mitochondrial (mt) and cytoplasmic (cy) tRNAs in vivo. Depletion of Nfs1p resulted in an immediate impairment of the 2-thio-modification of 5-carboxymethylaminomethyl-2-thiouridine at the wobble positions of mt-tRNA(UUU)(Lys) and mt-tRNA(UUG)(Gln). In addition, we observed a severe reduction in the 2-thio-modification of 5-methoxycarbonylmethyl-2-thiouridine (mcm(5)s(2)U) of cy-tRNA(UUU)(Lys2) and cy-tRNA(UUC)(Glu3), although the effect was somewhat delayed compared with that seen in mt-tRNAs. Mass spectrometry analysis revealed an increase in 5-methoxycarbonylmethyluridine concomitant with a decrease in mcm(5)s(2)U in cy-tRNAs that were prepared from Nfs1p-depleted cells. These results suggest that Nfs1p is involved in the 2-thio-modification of both 5-carboxymethylaminomethyl-2-thiouridine in mt-tRNAs and mcm(5)s(2)U in cy-tRNAs.

Blotting, Northern↗
PubMed1992

Absence of tRNA-guanine transglycosylase in a human colon adenocarcinoma cell line.

Queuosine (Q), found exclusively in the first position of the anticodons of tRNA(Asp), tRNA(Asn), tRNA(His) and tRNA(Tyr), is synthesized in eucaryotes by a base-for-base exchange of queuine, the base of Q, for guanine at tRNA position 34. This reaction is catalyzed by the enzyme tRNA-guanine transglycosylase (EC 2.4.2.29). We measured the specific release of queuine from Q-5'-phosphate (queuine salvage) and the extent of tRNA Q modification in 6 human tumors carried as xenografts in immune-deprived mice. Q-deficient tRNA was found in 3 of the tumors but it did not correlate with diminished queuine salvage. The low tRNA Q content of one tumor, the HxGC3 colon adenocarcinoma, prompted us to examine a HxGC3-derived cell line, GC3/M. GC3/M completely lacks Q in its tRNA and measurable tRNA-guanine transglycosylase activity; the first example of a higher eucaryotic cell which lacks this enzyme. Exposure of GC3/M cells to 5-azacytidine induces the transient appearance of Q-positive tRNA. This result suggests that at least one allele of the transglycosylase gene in GC3/M cells may have been inactivated by DNA methylation. In clinical samples, we found Q-deficient tRNA in 10 of 46 solid tumors, including 2 of 13 colonic carcinomas.

Adenocarcinoma↗
PubMed1995

Rp-phosphorothioate modifications in RNase P RNA that interfere with tRNA binding.

We have used Rp-phosphorothioate modifications and a binding interference assay to analyse the role of phosphate oxygens in tRNA recognition by Escherichia coli ribonuclease P (RNase P) RNA. Total (100%) Rp-phosphorothioate modification at A, C or G positions of RNase P RNA strongly impaired tRNA binding and pre-tRNA processing, while effects were less pronounced at U positions. Partially modified E. coli RNase P RNAs were separated into tRNA binding and non-binding fractions by gel retardation. Rp-phosphorothioate modifications that interfered with tRNA binding were found 5' of nucleotides A67, G68, U69, C70, C71, G72, A130, A132, A248, A249, G300, A317, A330, A352, C353 and C354. Manganese rescue at positions U69, C70, A130 and A132 identified, for the first time, sites of direct metal ion coordination in RNase P RNA. Most sites of interference are at strongly conserved nucleotides and nine reside within a long-range base-pairing interaction present in all known RNase P RNAs. In contrast to RNase P RNA, 100% Rp-phosphorothioate substitutions in tRNA showed only moderate effects on binding to RNase P RNAs from E. coli, Bacillus subtilis and Chromatium vinosum, suggesting that pro-Rp phosphate oxygens of mature tRNA contribute relatively little to the formation of the tRNA-RNase P RNA complex.

Base Sequence↗
PubMed1994

Detection of human tRNAs with antisense oligonucleotides.

Regulated expression and modification of tRNA isoacceptors may play an important role in the control of gene expression during such processes as differentiation and immune activation. However, the development of techniques for the identification and quantitation of multiple tRNA isoaccepting species has been hindered by the relative physicochemical similarity among individual isoacceptors and their high degree of post-transcriptional modification. We have used antisense DNA oligonucleotides derived from the T stem to acceptor stem region of six human tRNAs and one murine tRNA to detect individual tRNA isoacceptors in slot blots, Northern blots, and dot blots of human tRNA. This hybridization protocol was used in combination with tRNA fractionation by electrophoresis on a partially denaturing gel by reversed-phase low pressure chromatography and reversed-phase HPLC to identify multiple tRNA isoacceptors in a single sample of tRNA. Using this technique, it should be possible to monitor changes in the cellular tRNA repertoire that may be involved in the regulation of gene expression.

Amino Acid Sequence↗
PubMed1997

The effect of pseudouridine and pH on the structure and dynamics of the anticodon stem-loop of tRNA(Lys,3).

The anticodon stem-loop hairpin of tRNA(Lys,3) was synthesized and the solution structure determined by NMR spectroscopy. The hairpin is thermodynamically stabilized by pseudouridine as determined by UV Tm measurements, and the local loop structure is stabilized with base-stacking of the nucleosides in the anticodon loop 5' of the psi 39 nucleoside modification. The tRNA(Lys,3) hairpin also contains an A(+)-C base-pair that effectively reduces the size of the normal 7 nucleotide anticodon loop to 5 nucleotides and induces a change in the loop backbone conformation. The stabilizing effects of the A(+)-C base-pair and pseudouridine are only partially additive, suggesting that the conformational changes caused by each modification are not completely compatible. The structure of the anticodon loop is distinctly different from that seen for other tRNAs exemplified by tRNA(Phe), suggesting that the full complement of modified nucleosides present in tRNA(Lys,3) should significantly change the structure compared to the unmodified tRNA anticodon loop. The conformation of the loop has important implications for the role of nucleoside modification in codon-anticodon recognition and for utilization of tRNA(Lys,3) by HIV-1 as the natural reverse transcriptase primer.

Anticodon↗
PubMedDate not supplied

Problems of translating heterologous genes in expression systems: the role of tRNA.

tRNA can have large effects on the expression and overexpression of heterologous genes in microbial expression systems through reduced translation and errors in amino acid sequences of protein products. Examples are given of large effects on gene expression related to tRNA content and to tRNA base modifications, both of which differ in heterologous expression systems compared to the cells from which the genes originally came. tRNA should be of greater concern in the expression of heterologous genes.

Animals↗
PubMed1983

Processing of tRNA is accomplished by a high-molecular-weight enzyme complex.

An enzyme complex is a multifunctional catalytic unit that efficiently associates substrates with functionally related enzymes. The enzyme complex provides for the cellular regulation of enzymatic activities by physical interaction of the proteins with each other and by prior alteration of one enzyme's substrate by a related enzyme. Such regulatory abilities may go awry in neoplasia. Components of the protein biosynthetic machinery, such as aminoacyl-tRNA synthetases, have been thought to exist freely in the cytoplasm. However, high-molecular-weight enzyme complexes with aminoacyl-tRNA synthetase activities have been found in mammalian cells. We have been the first to report that the mammalian cell enzymes responsible for modification of tRNA occur in enzyme complexes (molecular weight 900000 daltons) associated with aminoacyl-tRNA synthetases and that the activities of these enzymes differ in normal and leukemic cells. Thus the enzymes responsible for the methylation of tRNA occur in enzyme complexes that provide efficient maturation of tRNA and possible regulation of protein synthesis. In FLC cells a unique enzyme complex composed of tRNA-methyltransferase and aminoacyl-tRNA synthetase activities has also been shown to contain a specific ribonuclease activity and a cysteine-tRNA sulfurtransferase activity. Sulfurtransferase activity has been characterized and optimized for its tRNA and cysteine substrates and mercaptoethanol and cation cofactors. Abnormal activity of this enzyme during neoplasia could result in improper acylation of tRNA and/or infidelity of coding by tRNA. Specific RNase is important in the sizing of percursor tRNA into mature tRNA. Results showed that this sizing was dependent upon the presence of the enzyme complex and the length of the incubation time. Many of the 20 aminoacyl-tRNA synthetases are also found in the complex. Electron microscopy has verified the subunit nature of the complex, seen previously by density gradient centrifugation and gel filtration. Three subunits, each of 300 000 daltons, comprise a complex approximately 200 A in diameter.

Amino Acyl-tRNA Synthetases↗
PubMed1996

Modulation of queuine uptake and incorporation into tRNA by protein kinase C and protein phosphatase.

It has been suggested that the rate of queuine uptake into cultured human fibroblasts is controlled by phosphorylation levels within the cell. We show that the uptake of queuine is stimulated by activators of protein kinase C (PKC) and inhibitors of protein phosphatase; while inhibitors of PKC, and down-regulation of PKC by chronic exposure to phorbol esters inhibit the uptake of queuine into cultured human fibroblasts. Activators of cAMP- and cGMP-dependent kinases exert no effect on the uptake of queuine into fibroblast cell cultures. These studies suggest that PKC directly supports the activity of the queuine uptake mechanism, and that protein phosphatase activity in the cell acts to reverse this. Regardless of the modulation of uptake rate, the level of intracellular queuine base saturates in 6 h. However, there is still an effect on the incorporation rate of queuine into tRNA of fibroblast cultures even after 24 h. We now show that the incorporation of queuine into tRNA in cultured human fibroblasts by tRNA-guanine ribosyltransferase (TGRase) is also stimulated by activators of PKC and inhibitors of protein phosphatase; while inhibitors of PKC decrease the activity of this enzyme. These studies suggest that PKC supports both the cellular transport of queuine and the activity of TGRase in cultured human fibroblasts, and that protein phosphatase activity in fibroblasts acts to reverse this phenomenon. A kinase-phosphatase control system, that is common to controlling both intracellular signal transduction and many enzyme systems, appears to be controlling the availability of the queuine substrate and the mechanism for its incorporation into tRNA. Since hypomodification of transfer RNA with queuine is commonly observed in undifferentiated, rapidly growing and neoplastically transformed cells, phosphorylation of the queuine modification system may be a critical regulatory mechanism for the modification of tRNA and subsequent control of cell growth and differentiation.

Biological Transport↗
PubMed1993

5-Methylcytidine is required for cooperative binding of Mg2+ and a conformational transition at the anticodon stem-loop of yeast phenylalanine tRNA.

The role of modified nucleosides in tRNA structure and ion binding has been investigated with chemically synthesized RNAs corresponding to the yeast tRNA(Phe) anticodon stem and loop (tRNA(ACPhe). Incorporation of d(m5C) at position 14 of the stem of tRNA(ACPhe)-d(m5C14), CCAGACUGAAGAU-d(m5C14)-UGG, analogous to m5C40 in native tRNA(Phe), introduced a strong Mg2+ binding at a site distant from the m5C. A Mg(2+)-induced structural transition, detected by circular dichroism spectroscopy, was similar to that observed for the DNA analog of tRNA(ACPhe) (Guenther et al., 1992; Dao et al., 1992). In contrast, Mg2+ had little effect on unmodified tRNA(ACPhe)-rC14 or tRNA(ACPhe)-d(C14). Modified tRNA(ACPhe)-d(m5C14) bound two Mg2+ ions, and the binding was cooperative. The dissociation constant of the two Mg2+ ions from tRNA(ACPhe)-d(m5C14), 2.5 x 10(-9) M2, is the result of an RNA structure significantly stabilized by Mg2+ binding, delta G = -11.7 kcal/mol. The tRNA(ACPhe)-d(m5C14) structure, investigated by 1H NMR, had a double stranded stem of five base pairs and two additional base pairs across what was a seven membered loop in the unmodified tRNA(Phe)AC. Methylation of cytidine in the yeast tRNA(ACPhe) enables the molecule to form more than one conformation through a process regulated by Mg2+ concentration. Thus, the simplest of posttranscriptional modifications of tRNA, a methylation, is involved in a somewhat distant, internal-site Mg2+ binding and stabilization of tRNA structure, especially that of the anticodon stem and loop.

Anticodon↗
PubMed1991

[Chemical modification of lysine residues in tyrosyl-tRNA-synthetase from cattle liver using pyridoxal-5'-phosphate].

Chemical modification of lysine residues of eukaryotic tyrosyl-tRNA synthetase was studied. It was shown that only four out of 22 lysine residues per enzyme dimer could be modified with pyridoxal-5'-phosphate. This modification led to the inactivation of tRNATyr aminoacylation by more than 90% but did not practically affect the rate of ATP-[32P]pyrophosphate exchange. Low molecular weight substrates (ATP, ATP-tyrosine) weakly protected the enzyme from inactivation, whereas tRNATyr afforded a much more effective protection. It was supposed that lysine residues of tyrosyl-tRNA synthetase can be involved in the interaction with tRNATyr.

Animals↗