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PubMedDate not supplied

Microarray expression profiling identifies early signaling transcripts associated with 6-OHDA-induced dopaminergic cell death.

The parkinsonian mimetic 6-hydroxydopamine (6-OHDA) has been shown to cause transcriptional changes associated with cellular stress and the unfolded protein response. As these cellular sequelae depend on upstream signaling events, the present study used functional genomics and proteomic approaches to aid in deciphering toxin-mediated regulatory pathways. Microarray analysis of RNA collected from multiple time points following 6-OHDA treatment was combined with data mining and clustering techniques to identify distinct functional subgroups of genes. Notably, stress-induced transcription factors such as ATF3, ATF4, CHOP, and C/EBP beta were robustly up-regulated, yet exhibited unique kinetic patterns. Genes involved in the synthesis and modification of proteins (various tRNA synthetases), protein degradation (e.g., ubiquitin, Herpud1, Sqstm1), and oxidative stress (Hmox1, Por) could be subgrouped into distinct kinetic profiles as well. Realtime PCR and/or two-dimensional electrophoresis combined with western blotting validated data derived from microarray analyses. Taken together, these data support the notion that oxidative stress and protein dysfunction play a role in Parkinson's disease, as well as provide a time course for many of the molecular events associated with 6-OHDA neurotoxicity.

Animals↗
PubMed2004

Structural significances of pseudouridination and methylation of uridine residues in tRNA: X-ray analyses of DNA dodecamers containing uridine and pseudouridine.

Pseudouridine (psi) and 5-methyluridine (T) residues found in tRNA are prepared through modification of the uridine residues by specific enzymes just after transcription. On the other hands, thymidine residues in DNA are incorporated using dTTP which is derived from UTP before replication. In order to investigate the necessity of such modification and the structural properties of these residues, we have determined two X-ray structures of DNA dodecamers containing dU or d psi, and compared with that containing dT. There is found remarkable difference in arrangement of water molecules hydrogen-bonded to these residues, which form a Watson-Crick type base pair. The psi residue stabilizes the phosphate-backbone conformation of the phosphate group via water mediated hydrogen bond networks, while dT residues seem to prevent from attacking of water molecules.

Crystallography, X-Ray↗
PubMed2004

The natural intron sequence of human tyrosine pre-transfer RNA is not a temporal stabilizer for cloverleaf structure.

We have developed the hyperprocessing technique to evaluate the stability of the cloverleaf shape of pre-transfer RNA (pre-tRNA). Application of this strategy to hyperprocessible human tyrosine pre-tRNA indicated that the natural intron sequence did not contribute to stabilization of the cloverleaf shape of this pre-tRNA, while the artificial intron with elongated anticodon-stem completely inhibited hyperprocessing of it. Our data suggested that the contemporary intron sequence may be a vestige of the ancient pre-biotic world, but not has been retained as a temporal stabilizer of the pre-tRNA before the base modifications.

Base Sequence↗
PubMed2005

Statin-associated neuromyotoxicity.

The sequelae of cardiovascular disease contribute significantly to morbidity and mortality in developed nations. As a class, the statins have been shown to measurably reduce the burden of atherosclerotic illness. However, muscle- and, more recently, nerve-related toxicity have emerged as potential complications leading to treatment withdrawal. Generally, the myopathic signs and symptoms of tenderness, myalgias, cramping and elevated serum creatine kinase (CK) activity are fully reversible after drug discontinuation. Growing evidence suggests that latent or previously minimal symptomatic muscle disease may predispose to the development of myopathy. Less information is available regarding the natural history of the sensorimotor neuropathy, but it appears to be less reversible if large fiber function is clinically manifest. Pathophysiologic clues regarding the potential causes of statin myopathy with or without neuropathy are discussed with particular attention paid to the implications of disrupted mevalonate metabolism. For example, secondary defects in isoprenoid biosynthesis are expected to impair the production of a variety of intermediaries such as dolichols, which are crucial for N-linked glycosylation; geranylgeranyl pyrophosphate, which is necessary for coenzyme Q(10) and G-protein synthesis; farnesyl-pyrophosphate, which facilitates the endoproteolytic cleavage and maturation of prelamin A and modifies B-type lamins and G-proteins; and isopentenylpyrophosphate, which is involved in a nucleoside modification of selenocysteinyl-tRNA and thus indirectly related to the synthesis of all selenoproteins (approximately 35). The nature of statin neuromyotoxicity remains unresolved; however, investigating the cellular corollaries of deranged isoprenoid metabolism may uncover clues that lead to a more complete understanding of the elusive pathophysiology.

Acyl Coenzyme A↗
PubMedDate not supplied

[The effect of modification of nucleotide-37 on the interaction of aminoacyl-tRNA with the A-site of the 70S ribosome].

To estimate the effect of modified nucleotide-37, the interaction of two yeast aminoacyl-tRNAs (Phe-tRNAK+YPhe and Phe-tRNAK-YPhe) with the A site of complex [70S.poly(U).deacylated tRNA(Phe) in the P site] was assayed at 0-20 degrees C. As comparisons with native Phe-tRNAK+YPhe showed, removal of the Y base decreased the association constant of Phe-tRNAK-YPhe and the complex by an order of magnitude at any temperature, and increased the enthalpy of their interaction by 23 kJ/mol. When the Y base was present in the anticodon loop of deacylated tRNA(Phe) bound to the P site of the 70S ribosome, twice higher affinity for the A site was observed for Phe-tRNAK-YPhe but not for Phe-tRNAK+YPhe. Thus, the modified nucleotide 3' of the Phe-tRNA(Phe) anticodon stabilized the codon-anticodon interaction both in the A and in the P sites of the 70S ribosome.

Anticodon↗
PubMed2003

The human TruB family of pseudouridine synthase genes, including the Dyskeratosis Congenita 1 gene and the novel member TRUB1.

A novel human gene denominated TruB pseudouridine (psi) synthase homolog 1 (E. coli) (approved symbol, TRUB1) has been identified and characterized. Spanning approximately 40 kb on chromosome 10 and including 8 exons, TRUB1 is the first described human ortholog of bacterial TruB/psi55, a gene involved in tRNA pseudouridinilation. TRUB1 gene encodes a 349-amino acid product, with a VFAVHKPKGPTSA box in positions 71-83 corresponding to motif I of the TruB family (probably involved in conserving protein structure). The TruB domain of TRUB1 lies between W104 and I255, and contains another short motif, GGTLDS AARGVLVV, including the highly conserved D residue that characterizes motif II (involved in uridine recognition and in catalytic function of psi synthases). Northern blot analysis revealed that TRUB1 mRNA is widely expressed in various human tissues (especially heart, skeletal muscle and liver). Phylogenetic analysis of the TruB domain revealed another human gene (approved symbol TRUB2) encoding a conserved TruB domain, located on human chromosome 9. Thus, the human TruB family includes at least three members: i.e. DKC1 (previously identified), TRUB1 and TRUB2. The TRUB1 and TRUB2 products could be the hitherto unidentified human tRNA psi synthases. Although TRUB1 is not highly similar to DKC1/dyskerin (whose mutations cause X-linked dyskeratosis congenita) and putatively affects tRNA rather than rRNA modification, it is the most similar human protein to dyskerin. Study of TRUB1 (and TRUB2) should facilitate understanding of the molecular mechanisms of RNA modification and the involvement of psi synthases in human pathology, including dyskeratosis-like diseases.

3' Untranslated Regions↗
PubMed2005

Statin-associated neuromyotoxicity.

The sequelae of cardiovascular disease contribute significantly to morbidity and mortality in developed nations. As a class, the statins have been shown to measurably reduce the burden of atherosclerotic illness. However, muscle- and, more recently, nerve-related toxicity have emerged as potential complications leading to treatment withdrawal. Generally, the myopathic signs and symptoms of tenderness, myalgias, cramping and elevated serum creatine kinase (CK) activity are fully reversible after drug discontinuation. Growing evidence suggests that latent or previously minimal symptomatic muscle disease may predispose to the development of myopathy. Less information is available regarding the natural history of the sensorimotor neuropathy, but it appears to be less reversible if large fiber function is clinically manifest. Pathophysiologic clues regarding the potential causes of statin myopathy with or without neuropathy are discussed with particular attention paid to the implications of disrupted mevalonate metabolism. For example, secondary defects in isoprenoid biosynthesis are expected to impair the production of a variety of intermediaries such as dolichols, which are crucial for N-linked glycosylation; geranylgeranyl pyrophosphate, which is necessary for coenzyme Q10 and G-protein synthesis; farnesyl-pyrophosphate, which facilitates the endoproteolytic cleavage and maturation of prelamin A and modifies B-type lamins and G-proteins; and isopentenylpyrophosphate, which is involved in a nucleoside modification of selenocysteinyl-tRNA and thus indirectly related to the synthesis of all selenoproteins (estimated at 35). The nature of statin neuromyotoxicity remains unresolved; however, investigating the cellular corollaries of deranged isoprenoid metabolism may uncover clues that lead to a more complete understanding of the elusive pathophysiology.

Creatine Kinase↗
PubMed1985

Processing of precursor tRNAs in Drosophila. Processing of the 3' end involves an endonucleolytic cleavage and occurs after 5' end maturation.

Transfer RNA biosynthesis is a complex process which includes size trimming and nucleotide modification of an initial tRNA precursor. We have examined the temporal order and the nature of tRNA processing events in a Drosophila in vitro transcription/processing system using Drosophila tRNA genes as templates. RNA sequence analysis of processing products indicates that processing at both 5' and 3' ends occurs by endonucleolytic cleavage. The time course of processing of an initial tRNA precursor to mature tRNA reveals that trimming at the 5' end precedes 3' end maturation.

Animals↗
PubMed1977

Preparation of Escherichia coli tRNAs terminating of modified nucleosides by the use of CTP(ATP):tRNA nucleotidyltransferase and polynucleotide phosphorylase.

Two procedures were investigated for the modification of tRNAs at the 3'-terminal nucleoside. The first involved the incubation of an enzymatically abreviated tRNA (tRNA-C-COH) with appropriate nucleoside triphosphates in the presence of CTP(ATP):tRNA nucleotidyltransferase from Escherichia coli and yeast. The E. coli enzyme did not utilize 2'- or 3'-deoxyadenosine 5'-triphosphate as substrates, but affected incorporation of the 2'- and 3'-O-methyladenosine triphosphates onto tRNA-C-Cou to the extent of 30 and 37%, respectively. Although incorporation of the deoxynucleotides could not be effected using the E. coli enzyme, yeast CTP(ATP:tRNA nucleotidyltransferase produced the desired tRNAs in yields of 45-65%. The second modification procedure involved incubation of tRNA-C-COH with (appropriately blocked) nucleoside diphosphates in the presence of polynucleotide phosphorylase. This procedure afforded the tRNAs terminating in 2'- and 3'-deoxyadenosine in yields of 4% (and the yield of the former was increased to 36% when the incubation was carried out in the presence of 20% methanol). The yields of tRNAs terminating in 2'- and 3'-O-methyladenosing produced by this procedure were 55 and 17%, respectively. Because only single isomers of most of the tRNAs terminating in 2'- and 3'-deoxy- and O-methyladenosine are aminoacylated, attempts were made to obtain the other isomericaminoacyl-tRNA by enzymatic introduction of chemically preaminoacylated nucleotides onto tRNA-C-COH. Although incubation of tRNA-C-COH with three aminoacylated nucleoside 5'-triphosphates and E. coli CTP(ATP):tRNA nucleotidyltransferase did not result in production of the desired tRNAs to a detectable extent, incubation with 2'-deoxy-3'-O-L-phenylalanyladenosine 5'-diphosphate and polynucleotide phosphorylase afforded E. coli tRNA terminating with the corresponding aminoacylated deoxynucleoside.

Adenosine Diphosphate↗
PubMed1984

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.

Autoradiography↗
PubMed2003

Caenorhabditis elegans pseudouridine synthase 1 activity in vivo: tRNA is a substrate, but not U2 small nuclear RNA.

The formation of pseudouridine (Psi) from uridine is post-transcriptional and catalysed by pseudouridine synthases, several of which have been characterized from eukaryotes. Pseudouridine synthase 1 (Pus1p) has been well characterized from yeast and mice. In yeast, Pus1p has been shown to have dual substrate specificity, modifying uridines in tRNAs and at position 44 in U2 small nuclear RNA (U2 snRNA). In order to study the in vivo activity of a metazoan Pus1p, a knockout of the gene coding for the homologue of Pus1p in Caenorhabditis elegans was obtained. The deletion encompasses the first two putative exons and includes the essential aspartate that is required for activity in truA pseudouridine synthases. The locations of most modified nucleotides on small RNAs in C. elegans are not known, and the positions of Psi were determined on four tRNAs and U2 snRNA. The uridine at position 27 of tRNA(Val) (AAC), a putative Pus1p-modification site, was converted into Psi in the wild-type worms, but the tRNA(Val) (AAC) from mutant worms lacked the modification. Psi formation at positions 13, 32, 38 and 39, all of which should be modified by other pseudouridine synthases, was not affected by the loss of Pus1p. The absence of Pus1p in C. elegans had no effect on the modification of U2 snRNA in vivo, even though worm U2 snRNA has a Psi at position 45 (the equivalent of yeast U2 snRNA position 44) and at four other positions. This result was unexpected, given the known dual specificity of yeast Pus1p.

Animals↗
PubMed1999

The cleavage step of ribonuclease P catalysis is determined by ribozyme-substrate interactions both distal and proximal to the cleavage site.

The cleavage step of bacterial RNase P catalysis involves concentration-independent processes after the formation of the ribozyme-substrate complex that result in the breaking of a phosphodiester bond. The 2'OH group at the cleavage site of a pre-tRNA substrate is an important determinant in the cleavage step. We determined here that in contrast to a tRNA substrate, the 2'OH at the cleavage site of two in vitro selected substrates has no effect, whereas a 2'OH located adjacent to the cleavage site has a similarly large effect on the cleavage step. This result indicates that a unique 2'OH in the vicinity of the cleavage site interacts with the ribozyme to achieve the maximal efficiency of the cleavage step. Individual modifications in a pre-tRNA substrate that disrupt ES interactions proximal to the cleavage site generally have little effect on the usage of this unique 2'OH. Ribozyme modifications that delete the interactions involving the T stem-loop of the tRNA have a large effect on the usage of this unique 2'OH and also alter the location of this 2'OH. We propose a new ES complex prior to the bond-breaking step in the reaction scheme to explain these results. This second ES complex is in fast equilibrium with the initial ES complex formed by bimolecular collision. The ribozyme interaction with this unique 2'OH shifts the equilibrium in favor of the second ES complex. The formation of the second ES complex may require optimal geometry of the two independently folding domains of this ribozyme to precisely position crucial functional groups and Mg2+ ions in the active site. Such a domain geometry is significantly favored by the RNase P protein. In the absence of the protein, spatial rearrangement of these domains in the ES complex may be necessary.

Bacillus subtilis↗
PubMed1996

Defective transfer RNA-queuine modification in C3H10T1/2 murine fibroblasts transfected with oncogenic ras.

tRNA isoacceptors for aspartic acid, asparagine, histidine, and tyrosine are modified in the anticodon wobble position with the deazaguanine analogue queuine. Queuine modification is defective in many tumors and transformed cell lines, and the extent of hypomodification correlates with staging and outcome in numerous human tumors. The molecular role of queuine modification in normal cells and the mechanisms of queuine hypomodification in tumors are unknown. We have characterized nontransformed C3H10T1/2 murine fibroblasts (C3H) and their ras-transfected counterparts (RasC4) with respect to the causes and effects of queuine hypomodification. RasC4 cells are hypomodified for queuine compared with C3H cells, despite increase tRNA-guanine ribosyltansferase activity. Excess exogenous queuine can cause repletion of tRNA queuine levels in RasC4 cells. Queuine modification of both C3H and RasC4 cells can be decreased by treatment with 7-methylguanine. This treatment does not affect growth in monolayer culture but enhances anchorage-independent growth of RasC4 cells greatly. These cell lines may be useful systems for the study of queuine function in normal cells and the causes and consequences of hypomodification for queuine in tumors.

Animals↗
PubMed2002

Identification of specific HIV-1 reverse transcriptase contacts to the viral RNA:tRNA complex by mass spectrometry and a primary amine selective reagent.

We have devised a high-resolution protein footprinting methodology to dissect HIV-1 reverse transcriptase (RT) contacts to the viral RNA:tRNA complex. The experimental strategy included modification of surface-exposed lysines in RT and RT-viral RNA:tRNA complexes by the primary amine selective reagent NHS-biotin, SDSPAGE separation of p66 and p51 polypeptides, in gel proteolysis, and comparative mass spectrometric analysis of peptide fragments. The lysines modified in free RT but protected from biotinylation in the nucleoprotein complex were readily revealed by this approach. Results of a control experiment examining the RT-DNA:DNA complex were in excellent agreement with the crystal structure data on the identical complex. Probing the RT-viral RNA:tRNA complex revealed that a majority of protein contacts are located in the primer-template binding cleft in common with the RT-DNA:DNA and RT-RNA:DNA species. However, our footprinting data indicate that the p66 fingers subdomain makes additional contacts to the viral RNA:tRNA specific for this complex and not detected with DNA:DNA. The protein footprinting method described herein has a generic application for high-resolution solution structural studies of multiprotein-nucleic acid contacts.

Amino Acid Sequence↗
PubMed1998

23S rRNA positions essential for tRNA binding in ribosomal functional sites.

rRNA plays an important role in function of peptidyl transferase, the catalytic center of the ribosome responsible for the peptide bond formation. Proper placement of the peptidyl transferase substrates, peptidyl-tRNA and aminoacyl-tRNA, is essential for catalysis of the transpeptidation reaction and protein synthesis. In this report, we define a small set of rRNA nucleotides that are most likely directly involved in binding of tRNA in the functional sites of the large ribosomal subunit. By binding biotinylated tRNA substrates to randomly modified large ribosomal subunits from Escherichia coli and capturing resulting complexes on the avidin resin, we identified four nucleotides in the large ribosomal subunit rRNA (positions G2252, A2451, U2506, and U2585) whose modifications prevent binding of a peptidyl-tRNA analog in the P site and one residue (U2555) whose modification interferes with transfer of peptidyl moiety to puromycin. These nucleotides represent a subset of positions protected by tRNA analogs from chemical modification and significantly narrow the number of 23S rRNA nucleotides that may be directly involved in tRNA binding in the ribosomal functional sites.

Binding Sites↗
PubMed1984

The effect of tRNA binding on the structure of 5 S RNA in Escherichia coli. A chemical modification study.

The structure of 5 S RNA within the 70 S ribosome from Escherichia coli was studied using the chemical reagent kethoxal (alpha-keto-beta-ethoxybutyraldehyde) to modify accessible guanosines. The modification pattern of 5 S RNA from free 70 S ribosomes was compared with that of poly(U) programmed ribosomes where tRNA had been bound to both the A- and P-sites. Binding to the ribosomal A-site was achieved enzymatically using the elongation factor Tu and GTP in the presence of deacylated tRNA which blocks the ribosomal P-site. Modified guanosines were identified after partial RNase T1 hydrolysis and separation of the hydrolysis products on sequencing gels. Binding of tRNA to the ribosome leads to a strong protection of 5 S RNA guanosine G-41 and to some degree G-44 from kethoxal modification. The limited RNase T1 hydrolysis pattern provides evidence for the existence of a 5 S RNA conformation different from the known 5 S RNA A- and B-forms which are characterized by their gel electrophoretic mobility. The importance of 5 S RNA for the binding of tRNA to the ribosome is discussed.

Base Sequence↗
PubMed2000

Effects of anticodon 2'-O-methylations on tRNA codon recognition in an Escherichia coli cell-free translation.

The methylation of 2'-hydroxyl groups is one of the most common posttranscriptional modifications of naturally occurring stable RNA molecules. Some tRNA species have a 2'-O-methyl nucleoside at the first position of the anticodon, and it was suggested that this modification stabilizes the codon-anticodon duplex. However, no tRNA species have been found to have the modification at the second or third position of the anticodon. In the present study, we measured the effects of anticodon 2'-O-methylation on the codon-reading efficiencies of the anticodon variants of the unmodified forms of Escherichia coli tRNA1(Ser), using a cell-free protein synthesis assay. The modification of C in the first position of the anticodon into 2'-O-methylcytidine increased the efficiency of reading the G-ending codon. On the other hand, the modifications of the second and/or third positions were detrimental to the codon-reading activity. Thus, 2'-hydroxyl groups at the second and third positions of the anticodon may have some role in the translation reaction, and this may be the reason why 2'-O-methyl nucleosides are not found in these positions within natural tRNA species.

Amino Acid Sequence↗
PubMed1984

A modified nucleotide in tRNA as a possible regulator of aerobiosis: synthesis of cis-2-methyl-thioribosylzeatin in the tRNA of Salmonella.

The state of modification of the adenosine residue (A37), found adjacent to the anticodon in tRNAs that recognize codons beginning with U, varies in Salmonella bacteria grown under different physiological conditions. In aerobically grown bacteria, these tRNAs contain ms2io6A and in bacteria grown anaerobically they contain its precursor, ms2i6A. The hydroxylation of the isopentenyl (i6-) side chain of ms2i6A does not occur in the absence of oxygen. When the bacteria are grown under iron or cysteine limitation the tRNAs contain predominantly i6A, rather than ms2i6A, ms2io6A, or io6A. The bacteria do not methylthiolate (ms2-) the i6A under these conditions. A Salmonella miaA mutant lacking the isopentenylation enzyme contains an A37 rather than any of the modified forms. Some of the biosynthetic pathways of the amino acids corresponding to ms2i6A containing tRNAs (phe, tyr, trp, ser, leu, cys) are known to have altered regulation depending on the state of modification of nucleoside A37. This regulation appears to be effected through attenuation. We hypothesize that these varying states of modification are related to electron-acceptor pathways in anaerobic or aerobic growth. The role of ms2io6-adenine (the cytokinin hormone in plants) and i6-adenine (an activator of the cell cycle in animal cells) is discussed as related to the role of modifying enzymes in regulation.

Adenosine↗