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PubMed1995

The role of nucleotide modifications in the yeast mitochondrial ribosome.

Post-transcriptionally modified nucleotides in ribosomal RNA, and the modifying enzymes themselves, could influence the assembly, structure, and function of the ribosome. The PET56 gene in yeast encodes the enzyme responsible for formation of 2'-O-methylguanosine at a specific nucleotide in the peptidyl transferase center of the mitochondrial large subunit ribosomal RNA. While PET56 is normally essential for the formation of functional mitochondrial ribosomes, extragenic mutations have been obtained that suppress, albeit weakly, pet56 loss-of-function mutations. Thus neither the Pet56p-catalyzed ribose methylation nor the Pet56 protein itself is absolutely required for the synthesis of a functional ribosome.

Genes, Fungal↗
PubMed2005

Precursor complex structure of pseudouridine synthase TruB suggests coupling of active site perturbations to an RNA-sequestering peripheral protein domain.

The pseudouridine synthase TruB is responsible for the universally conserved post-transcriptional modification of residue 55 of elongator tRNAs. In addition to the active site, the "thumb", a peripheral domain unique to the TruB family of enzymes, makes extensive interactions with the substrate. To coordinate RNA binding and release with catalysis, the thumb may be able to sense progress of the reaction in the active site. To establish whether there is a structural correlate of communication between the active site and the RNA-sequestering thumb, we have solved the structure of a catalytically inactive point mutant of TruB in complex with a substrate RNA, and compared it to the previously determined structure of an active TruB bound to a reaction product. Superposition of the two structures shows that they are extremely similar, except in the active site and, intriguingly, in the relative position of the thumb. Because the two structures were solved using isomorphous crystals, and because the thumb is very well ordered in both structures, the displacement of the thumb we observe likely reflects preferential propagation of active site perturbations to this RNA-binding domain. One of the interactions between the active site and the thumb involves an active site residue whose hydrogen-bonding status changes during the reaction. This may allow the peripheral RNA-binding domain to monitor progress of the pseudouridylation reaction.

Binding Sites↗
PubMed1998

DNA ribonucleases that are active against intracellular hepatitis B viral RNA targets.

DNA ribonucleases directed against direct repeat 1 (DR1) and polyadenylation signal regions of hepatitis B virus (HBV) messages were prepared with phosphorothioate modifications and varying arm lengths. DNA ribonucleases modified throughout the entire molecule and in the target binding arms were completely protected from degradation after incubation with serum. DNA ribonuclease modified only at the 5' and 3' termini remained 92.9% intact after incubation. Molecules with no modification were degraded to 67.6% under the same conditions. However, modification of the entire molecule and in the recognition arms resulted in 99.8% and 98.4% inactivation of cleavage activity, respectively. Modification of only the termini resulted in retention of 20% to 40% of original activity. Lengthening each terminally modified arm from 9 to 11 nucleotides increased cleavage efficiency almost 10-fold. In Huh 7 cells, DR1-directed DNA ribonucleases with terminal modifications significantly suppressed HBV-luciferase fusion gene expression up to 48% of control. In contrast, DNA ribonucleases had no effect on a control construct lacking any HBV target sequences. Moreover, inactivated mutant and HCV-directed DNA ribonucleases had no significant effects on the HBV target. We conclude that resistance of DNA ribonucleases to degradation can be enhanced through phosphorothioate modification. Cleavage activity can be retained by limiting modification to the termini and lengthening the recognition arms. Such DNA ribonucleases can be made to specifically cleave target HBV RNA and substantially inhibit intracellular viral gene expression.

Base Sequence↗
PubMed1993

Complex transcription of an operon encoding the SalI restriction-modification system of Streptomyces albus G.

High-resolution S1 nuclease mapping of mRNA synthesised in vivo, in vitro run-off transcription with RNA polymerase from Streptomyces lividans and gene fusions were used to analyse the transcriptional organization of the SalI restriction-modification system of Streptomyces albus G. The salIR and salIM genes that encode the restriction endonuclease and its cognate methyltransferase constitute an operon which is mainly transcribed from sal-pR1, a promoter located immediately upstream of salIR, with two possible minor promoters further upstream. Another promoter, sal-pM, is within the 3' end of the salIR coding region, and allows expression of the modification gene in the absence of sal-pR1. The sal-pM promoter might be involved in the establishment of modification prior to restriction endonuclease activity. Sequences upstream of the apparent transcriptional start sites for sal-pR1 and sal-pM show similarity with the -10 region of typical vegetatively expressed eubacterial promoters, but appropriately centered -35 regions are absent.

Base Sequence↗
PubMed1996

The canonical GU dinucleotide at the 5' splice site is recognized by p220 of the U5 snRNP within the spliceosome.

Specific recognition of the 5' splice site (5'SS) by the spliceosome components was studied using a simple in vitro system in which a short 5'SS RNA oligonucleotide specifically induces the assembly of snRNP particles into spliceosome-like complexes and actively participates in a trans-splicing reaction. Short-range cross-liking demonstrates that a U5 snRNP protein component, p220 (the human analogue of the yeast Prp8) specifically interacts with the invariant GU dinucleotide at the 5' end of the intron. The GU:p220 interaction can be detected in the functional splicing complex B. Although p220 has been known to contact several nucleotides around the 5' splice junction, the p220:GU dinucleotide interaction described here is remarkably specific. Consistent with the high conservation of the GU, even minor modifications of this element affect recognition of the 5'SS RNA by p220. Substitution of uridine at the GU with base analogues containing a large methyl or iodo group, but not a smaller flouro group at base position 5, interferes with association of 5'SS RNA with snRNP complexes and their functional participation in splicing.

Base Sequence↗
PubMed1999

Inhibition of transcription elongation in the HER-2/neu coding sequence by triplex-directed covalent modification of the template strand.

Triplex formation may be of potential utility to inhibit the expression of individual genes. We describe the formation of a triple helix in the coding sequence of the HER-2/neu gene. In vitro transcription analysis in the presence and absence of triplex formation demonstrates that an unmodified DNA triplex-forming oligonucleotide is incapable of inhibiting RNA polymerase elongation. Triplex formation by an oligonucleotide-psoralen conjugate was used to form a covalent photoadduct with a thymine on the nontemplate strand of the HER-2/neu gene. In the native HER-2/neu gene, covalent attachment of the triplex-forming oligonucleotide to the nontemplate strand did not prevent RNA polymerase elongation. Using HER-2/neu point mutants that would place the target thymine on the template strand, we demonstrated that covalent modification of the template strand was necessary to inhibit RNA polymerase elongation. Based on these data, we synthesized oligonucleotide-alkylator conjugates that would react with a specific guanine residue on the template strand of the HER-2/neu coding sequence. The oligonucleotide-alkylator conjugates inhibited transcription elongation by T7 RNA polymerase and eukaryotic RNA polymerase II from a HeLa nuclear extract. These studies demonstrate the successful application of triplex-forming oligonucleotide-alkylator conjugates to inhibit transcription elongation in the HER-2/neu gene, and show that covalent modification of the DNA strand used as the transcription template is necessary to prevent RNA polymerase elongation.

Binding Sites↗
PubMed2005

The Treacher Collins syndrome (TCOF1) gene product is involved in pre-rRNA methylation.

Treacher Collins syndrome (TCS) is characterized by defects in craniofacial development, which results from mutations in the TCOF1 gene. TCOF1 encodes the nucleolar phosphoprotein treacle, which interacts with upstream binding factor (UBF) and affects transcription of the ribosomal DNA gene. The present study shows participation of treacle in the 2'-O-methylation of pre-rRNA. Antisense-mediated down-regulation of treacle expression in Xenopus laevis oocytes reduced 2'-O-methylation of pre-rRNA. Analysis of RNA isolated from wild-type and Tcof1+/- heterozygous mice embryos from strains that exhibit a lethal phenotype showed significant reduction in 2'-O-methylation at nucleotide C463 of 18S rRNA. The level of pseudouridylation of U1642 of 18S rRNA from the same RNA samples was not affected suggesting specificity. There is no significant difference in rRNA methylation between wild-type and heterozygous embryos of DBA x BALB/c mice, which have no obvious craniofacial phenotype. The function of treacle in pre-rRNA methylation is most likely mediated by its direct physical interaction with NOP56, a component of the ribonucleoprotein methylation complex. Although treacle co-localizes with UBF throughout mitosis, it co-localizes with NOP56 and fibrillarin, a putative methyl transferase, only during telophase when rDNA gene transcription and pre-rRNA methylation are known to commence. These observations suggest that treacle might link RNA polymerase I-catalyzed transcription and post-transcriptional modification of pre-rRNA. We hypothesize that haploinsufficiency of treacle in TCS patients results in inhibition of production of properly modified mature rRNA in addition to inhibition of rDNA gene transcription, which consequently affects proliferation and proper differentiation of specific embryonic cells during development.

Animals↗
PubMed2003

Methylation of SPT5 regulates its interaction with RNA polymerase II and transcriptional elongation properties.

SPT5 and its binding partner SPT4 function in both positively and negatively regulating transcriptional elongation. The demonstration that SPT5 and RNA polymerase II are targets for phosphorylation by CDK9/cyclin T1 indicates that posttranslational modifications of these factors are important in regulating the elongation process. In this study, we utilized a biochemical approach to demonstrate that SPT5 was specifically associated with the protein arginine methyltransferases PRMT1 and PRMT5 and that SPT5 methylation regulated its interaction with RNA polymerase II. Specific arginine residues in SPT5 that are methylated by these enzymes were identified and demonstrated to be important in regulating its promoter association and subsequent effects on transcriptional elongation. These results suggest that methylation of SPT5 is an important posttranslational modification that is involved in regulating its transcriptional elongation properties in response to viral and cellular factors.

Arginine↗
PubMedDate not supplied

Biochemical modifications in the bursa of fabricius, thymus and adrenals of chickens injected with an anti-bursal serum.

Cornish-Rock chickens were given 0.3 ml anti-bursal serum in the pectoral muscle on the first day of life. Three days after serum administration, the modifications were evident both in the bursa of Fabricius and thymus (ponderal involution and decrease of RNA and glycogen contents). Pronounced thymo-bursal differences appeared at 13 days: in the bursa the values were normalized; in the thymus inverse modifications appeared as compared to the 3 days values. The modifications in the adrenals are in agreement with those in the lymph organs.

Adrenal Glands↗
PubMed1991

[IIa/IIo conversion of RNA polymerase II during heat shock].

Heat-shock treatment of cells activates a protein kinase which phosphorylates a heptapeptide analogous to the repeated motif of the C-terminal domain (CTD) of the large subunit of RNA polymerase II from mammalian cells. This is corroborated with a modification of the large subunit of this enzyme during thermal stress in HeLa cells. We have observed a shift from the IIa form (unphosphorylated) to the IIo form (phosphorylated) with a higher apparent molecular weight, during a heat-shock at 44, 45 or 46 degrees C and by a chemical stress induced by sodium arsenite. RNA polymerase II hyperphosphorylation, together with the activation of the heat-shock transcription factor, might contribute to the onset of the preferential transcription of heat-shock genes.

Arsenates↗
PubMed2001

Post-translational modification of the hepatitis C virus core protein by tissue transglutaminase.

The hepatitis C virus (HCV) core protein is a structural protein that packages the viral genomic RNA. In this study, we demonstrate that a stable core protein dimer could be produced in liver cells. The production of this protein could be enhanced by calphostin C and serum deprivation. This protein was determined to be the core protein dimer because of its reactivity with the anti-core antibody, its similar electrophoretic mobility compared with that of the core protein dimer generated by cross-linking with glutaraldehyde, and its increase in size by a hemagglutinin tag fused to the core protein sequence. This core protein dimer was highly stable and resistant to SDS and beta-mercaptoethanol. The enzyme that mediated the formation of this stable core protein dimer was determined to be the tissue transglutaminase (tTG) because, first, tTG could be activated by calphostin C and serum deprivation; second, the formation of this dimer was suppressed by monodansylcadaverine, a tTG inhibitor; and third, the core protein could be cross-linked by tTG in vitro. Thus, the HCV core protein represents the first known viral structural protein substrate of tTG. The post-translational modification by tTG reduced the RNA binding activity of the core protein, raising the possibility that tTG may regulate the biological functions of the HCV core protein.

Cell Line↗
PubMed2005

Kinase Cak1 functionally interacts with the PAF1 complex and phosphatase Ssu72 via kinases Ctk1 and Bur1.

Protein kinases orthologous with Cak1 of Saccharomyces cerevisiae (ScCak1) appear specific to ascomycetes. ScCak1 phosphorylates Cdc28, the cyclin-dependent kinase (CDK) governing the cell cycle, as well as Kin28, Bur1 and Ctk1, CDKs required for the transcription process performed by RNA polymerase II (RNA Pol II). Using genetic methods, we found that Cak1 genetically interacts with Paf1 and Ctr9, two components belonging to the PAF1 elongation complex needed for histone modifications, and with Ssu72, a protein phosphatase that dephosphorylates serine-5 phosphate in the RNA Pol II C-terminal domain. We present evidence suggesting that the interactions linking Cak1 with the PAF1 complex and with Ssu72 are not direct but mediated via Ctk1 and Bur1. We discuss the possibility that Ssu72 intervenes at the capping checkpoint step of the transcription cycle.

Base Sequence↗
PubMed2000

The human tRNA(m(2)(2)G(26))dimethyltransferase: functional expression and characterization of a cloned hTRM1 gene.

This paper presents the first example of a complete gene sequence coding for and expressing a biologically functional human tRNA methyltransferase: the hTRM1 gene product tRNA(m(2)(2)G)dimethyltransferase. We isolated a human cDNA (1980 bp) made from placental mRNA coding for the full-length (659 amino acids) human TRM1 polypeptide. The sequence was fairly similar to Saccharomyces cerevisiae Trm1p, to Caenorhabditis elegans TRM1p and to open reading frames (ORFs) found in mouse and a plant (Arabidopsis thaliana) DNA. The human TRM1 gene was expressed at low temperature in Escherichia coli as a functional recombinant protein, able to catalyze the formation of dimethylguanosine in E.coli tRNA in vivo. It targeted solely position G(26) in T7 transcribed spliced and unspliced human tRNA(Tyr) in vitro and in yeast trm1 mutant tRNA. Thus, the human TRM1 protein is a tRNA(m(2)(2)G(26))dimethyltransferase. Compared with yeast Trm1p, hTRM1p has a C-terminal protrusion of approximately 90 amino acids which shows similarities to a mouse protein related to RNA splicing. A deletion of these 90 C-terminal amino acids left the modification activity in vitro intact. Among point mutations in the hTRM1 gene, only those located in conserved regions of hTRM1p completely eliminated modification activity.

Cloning, Molecular↗
PubMed1999

AVANTI 1: randomized, double-blind trial to evaluate the efficacy and safety of zidovudine plus lamivudine versus zidovudine plus lamivudine plus loviride in HIV-infected antiretroviral-naive patients. AVANTI Study Group.

The objective of this randomized double-blind, placebo-controlled trial was to investigate the effect of combination antiretroviral therapy on plasma HIV-1 RNA as measured by HIV RNA PCR and to assess the safety and tolerability of such regimens. The trial was carried out in seven European countries, Australia and Canada and involved antiretroviral-naive patients (n = 106) with CD4 counts between 150-300 cells/mm3 (CDC group A) and 150-500 cells/mm3 (CDC group B/C). Patients were randomly assigned to zidovudine (200 mg three times daily) plus lamivudine (300 mg twice daily) or to zidovudine plus lamivudine plus loviride (100 mg three times daily) for 52 weeks. The main outcome measures were degree and duration of reduction of plasma HIV-1 RNA as measured by RNA PCR and the development of drug-related toxicities sufficiently severe to warrant dose modification, interruption or permanent discontinuation. A mild, though statistically significant difference in favour of zidovudine plus lamivudine plus loviride for log10 plasma HIV-1 RNA (P = 0.022), as compared to zidovudine plus lamivudine, was observed using area-under-the-curve minus baseline (AUCMB). An increase in CD4 cell count in the zidovudine plus lamivudine plus loviride group was observed with a median improvement of 124 cells/mm3 at week 52 compared with 70 cells/mm3 in the zidovudine plus lamivudine group (P = 0.06). Both treatment regimens were well tolerated.

Acetamides↗
PubMed1983

Carbodiimide inactivation of Escherichia coli RNA polymerase promoters on supercoiled simian virus 40 and ColE1 DNAs occurs by a one-hit process at salt concentrations in the physiological range.

A previous study (Hale, P., Woodward, R. W., and Lebowitz, J. (1980) Nature 284, 640-644) showed that Escherichia coli RNA polymerase promoters on superhelical SV40 DNA are highly selective targets for chemical modification by the water-soluble carbodiimide, N-cyclohexyl-N'-beta-(4-methylmorpholinium)ethyl carbodiimide (CMC). To extend the inactivation analysis of supercoiled DNAs, we determined the number and location of RNA polymerase binding sites on the supercoiled and linear forms of ColE1 DNA. We also determined the site distribution of [3H] CMC on the superhelical form. This information, coupled with per cent inhibition of transcription versus CMC-bound curves, allowed a test of the specificity of the CMC inactivation by the Poisson equation. Curves were obtained for supercoiled SV40 DNA modified at 0 and 100 mM NaCl (2 mM NaPi, pH 7.0) and for supercoiled ColE1 DNA modified at 0, 100, and 320 mM NaCl. For supercoiled SV40 DNA, these data, coupled to our knowledge of the number of RNA polymerase binding sites from the study cited above, revealed an excellent fit to a one-hit inactivation by the Poisson equation for DNA modified at 100 mM NaCl. For ColE1 DNA, we obtained an excellent fit to a Poisson distribution when supercoiled DNA was modified at 320 mM NaCl. The Poisson distribution can be applied to [3H] CMC restriction fragment data with equivalent results. These results suggest that promoter sites can be forced into different structural conformations with variable degrees of unpairing.

Affinity Labels↗
PubMed2006

Complete cap 4 formation is not required for viability in Trypanosoma brucei.

In kinetoplastids spliced leader (SL) RNA is trans-spliced onto the 5' ends of all nuclear mRNAs, providing a universal exon with a unique cap. Mature SL contains an m(7)G cap, ribose 2'-O methylations on the first four nucleotides, and base methylations on nucleotides 1 and 4 (AACU). This structure is referred to as cap 4. Mutagenized SL RNAs that exhibit reduced cap 4 are trans-spliced, but these mRNAs do not associate with polysomes, suggesting a direct role in translation for cap 4, the primary SL sequence, or both. To separate SL RNA sequence alterations from cap 4 maturation, we have examined two ribose 2'-O-methyltransferases in Trypanosoma brucei. Both enzymes fall into the Rossmann fold class of methyltransferases and model into a conserved structure based on vaccinia virus homolog VP39. Knockdown of the methyltransferases individually or in combination did not affect growth rates and suggests a temporal placement in the cap 4 formation cascade: TbMT417 modifies A(2) and is not required for subsequent steps; TbMT511 methylates C(3), without which U(4) methylations are reduced. Incomplete cap 4 maturation was reflected in substrate SL and mRNA populations. Recombinant methyltransferases bind to a methyl donor and show preference for m(7)G-capped RNAs in vitro. Both enzymes reside in the nucleoplasm. Based on the cap phenotype of substrate SL stranded in the cytosol, A(2), C(3), and U(4) methylations are added after nuclear reimport of Sm protein-complexed substrate SL RNA. As mature cap 4 is dispensable for translation, cap 1 modifications and/or SL sequences are implicated in ribosomal interaction.

Amino Acid Sequence↗
PubMed2006

Inducible yeast system for Viral RNA recombination reveals requirement for an RNA replication signal on both parental RNAs.

To facilitate RNA recombination studies, we tested whether Saccharomyces cerevisiae, which supports brome mosaic virus (BMV) replication, also supports BMV RNA recombination. Yeast strains expressing BMV RNA replication proteins 1a and 2a(pol) were engineered to transiently coexpress two independently inducible, overlapping, nonreplicating derivatives of BMV genomic RNA3. B3Delta3' lacked the coat protein gene and negative-strand RNA promoter. B3Delta5' lacked the positive-strand RNA promoter and had the coat gene replaced by the selectable URA3 gene. After 12 to 72 h of induction, B3Delta3' and B3Delta5' transcription was repressed and Ura(+) yeast cells were selected. All Ura(+) cells contained recombinant RNA3 replicons expressing URA3. Most replicons arose by intermolecular homologous recombination between B3Delta3' and B3Delta5'. Such recombinants were isolated only when 1a and 2a(pol) were expressed and after transient transcription of both B3Delta3' and B3Delta5', showing that recombination occurred at the RNA, not DNA, level. A minority of URA3-expressing replicons were derived from B3Delta5', independently of B3Delta3', by 5' truncation and modification, generating novel positive-strand promoters and demonstrating that BMV can give rise to subgenomic RNA replicons. Intermolecular B3Delta3'-B3Delta5' recombination occurred only when both parental RNAs bore a functional, cis-acting template recognition and recruitment element targeting viral RNAs to replication complexes. The results imply that recombination occurred in RNA replication complexes to which parental RNAs were independently recruited. Moreover, the ability to obtain intermolecular recombinants at precisely measurable, reproducible frequencies, to control genetic background and induction conditions, and other features of this system will facilitate further studies of virus and host functions in RNA recombination.

Base Sequence↗
PubMed2004

Structural rationalization of a large difference in RNA affinity despite a small difference in chemistry between two 2'-O-modified nucleic acid analogues.

Chemical modification of nucleic acids at the 2'-position of ribose has generated antisense oligonucleotides (AONs) with a range of desirable properties. Electron-withdrawing substituents such as 2'-O-[2-(methoxy)ethyl] (MOE) confer enhanced RNA affinity relative to that of DNA by conformationally preorganizing an AON for pairing with the RNA target and by improving backbone hydration. 2'-Substitution of the ribose has also been shown to increase nuclease resistance and cellular uptake via changes in lipophilicity. Interestingly, incorporation of either 2'-O-[2-(methylamino)-2-oxoethyl]- (NMA) or 2'-O-(N-methylcarbamate)-modified (NMC) residues into AONs has divergent effects on RNA affinity. Incorporation of 2'-O-NMA-T considerably improves RNA affinity while incorporation of 2'-O-NMC-T drastically reduces RNA affinity. Crystal structures at high resolution of A-form DNA duplexes containing either 2'-O-NMA-T or 2'-O-NMC-T shed light on the structural origins of the surprisingly large difference in stability given the relatively minor difference in chemistry between NMA and NMC. NMA substituents adopt an extended conformation and use either their carbonyl oxygen or amino nitrogen to trap water molecules between phosphate group and sugar. The conformational properties of NMA and the observed hydration patterns are reminiscent of those found in the structures of 2'-O-MOE-modified RNA. Conversely, the carbonyl oxygen of NMC and O2 of T are in close contact, providing evidence that an unfavorable electrostatic interaction and the absence of a stable water structure are the main reasons for the loss in thermodynamic stability as a result of incorporation of 2'-O-NMC-modified residues.

Biomimetic Materials↗