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PubMed1996

Pseudouridine synthases: four families of enzymes containing a putative uridine-binding motif also conserved in dUTPases and dCTP deaminases.

Using a combination of several methods for protein sequence comparison and motif analysis, it is shown that the four recently described pseudouridine syntheses with different specificities belong to four distinct families. Three of these families share two conserved motifs that are likely to be directly involved in catalysis. One of these motifs is detected also in two other families of enzymes that specifically bind uridine, namely deoxycitidine triphosphate deaminases and deoxyuridine triphosphatases. It is proposed that this motif is an essential part of the uridine-binding site. Two of the pseudouridine syntheses, one of which modifies the anticodon arm of tRNAs and the other is predicted to modify a portion of the large ribosomal subunit RNA belonging to the peptidyltransferase center, are encoded in all extensively sequenced genomes, including the 'minimal' genome of Mycoplasma genitalium. These particular RNA modifications and the respective enzymes are likely to be essential for the functioning of any cell.

Amino Acid Sequence↗

PubMed2025

Genome-Wide Association Study of Varenicline-Aided Smoking Cessation.

INTRODUCTION: Varenicline is an α4β2 nicotinic acetylcholine receptor partial agonist with the highest therapeutic efficacy of any pharmacological smoking cessation aid and a 12-month cessation rate of 26%. Genetic variation may be associated with varenicline response, but to date, no genome-wide association studies of varenicline response have been published. METHODS: In this study, we investigated the genetic contribution to varenicline effectiveness using two electronic health record-derived phenotypes. We defined short-term varenicline effectiveness (SVE) and long-term varenicline effectiveness (LVE) by assessing smoking status at 3 and 12 months, respectively, after initiating varenicline treatment. In Stage 1, comprising five European cohort studies, we tested genome-wide associations with SVE (1405 cases, 2074 controls) and LVE (1576 cases, 2555 controls), defining sentinel variants (the most strongly associated variant within 1 Mb) with p-value < 5 × 10-6 to follow up in Stage 2. In Stage 2, we tested association between sentinel variants and comparable smoking cessation endpoints in varenicline randomized controlled trials. We subsequently meta-analyzed Stages 1 and 2. RESULTS: No variants reached genome-wide significance in the meta-analysis. In Stage 1, 10 sentinel variants were associated with SVE and five with LVE at a suggestive significance threshold (p-value < 5 × 10-6); none of these sentinels were previously implicated in varenicline-aided smoking cessation or in genetic studies of smoking behavior. CONCLUSIONS: We provide initial insights into the biological underpinnings of varenicline-aided smoking cessation, through implicating genes involved in various processes, including gene expression, cilium assembly, and early-stage development. IMPLICATIONS: Leveraging electronic health records, we undertook the largest genetic study of varenicline-aided smoking cessation to date, and the only such study to test genome-wide associations. We showed distinct genetic variants associated (p-value < 5 × 10-6) with varenicline-aided smoking cessation which implicate diverse cellular functions, including transcriptional regulation, RNA modification, and cilium assembly. These provide insights which, if independently corroborated, will improve understanding of varenicline response. The growing availability of biobank resources with genetic and varenicline response data will provide future opportunities for larger studies using the approach we developed.

Humans↗

PubMed2002

A human mitochondrial transcription factor is related to RNA adenine methyltransferases and binds S-adenosylmethionine.

A critical step toward understanding mitochondrial genetics and its impact on human disease is to identify and characterize the full complement of nucleus-encoded factors required for mitochondrial gene expression and mitochondrial DNA (mtDNA) replication. Two factors required for transcription initiation from a human mitochondrial promoter are h-mtRNA polymerase and the DNA binding transcription factor, h-mtTFA. However, based on studies in model systems, the existence of a second human mitochondrial transcription factor has been postulated. Here we report the isolation of a cDNA encoding h-mtTFB, the human homolog of Saccharomyces cerevisiae mitochondrial transcription factor B (sc-mtTFB) and the first metazoan member of this class of transcription factors to which a gene has been assigned. Recombinant h-mtTFB is capable of binding mtDNA in a non-sequence-specific fashion and activates transcription from the human mitochondrial light-strand promoter in the presence of h-mtTFA in vitro. Remarkably, h-mtTFB and its fungal homologs are related in primary sequence to a superfamily of N6 adenine RNA methyltransferases. This observation, coupled with the ability of recombinant h-mtTFB to bind S-adenosylmethionine in vitro, suggests that a structural, and perhaps functional, relationship exists between this class of transcription factors and this family of RNA modification enzymes and that h-mtTFB may perform dual functions during mitochondrial gene expression.

Amino Acid Sequence↗

PubMed2005

A genomic-scale search for regulatory binding sites in the integration host factor regulon of Escherichia coli K12.

We examined general aspects of the DNA-protein interaction between the integration host factor (IHF) global regulator and its regulatory binding sites in the Escherichia coli K12 genome. Two models were developed with distinct weight matrices for the regulatory binding sites recognized by IHF. Using these matrices we performed a genome scale scan and built a set of computationally predicted binding sites for each of the models. The sites found by the model associated with repetitive sequences had a higher score in the sequence to matrix alignment. They were also more rare than the other sites. The sites not associated with repeats rapidly tended to become undistinguishable from the background as statistical stringency was relaxed. We compared our results to the known sites documented in RegulonDB and found new members of the IHF Regulon. The two models exhibit clearly distinct affinity patterns (scores in the sequence to matrix alignments and in the number of regulatory sites), as we vary the stringency of the statistical confidence parameters. We suggest that these differences may play an important role in the dynamics of the network. We concluded that IHF may regulate two genes encoding ATP-dependent RNA helicases. This interaction is not described in RegulonDB, even as a computational prediction. IHF may also regulate RNA modification processes.

Binding Sites↗

PubMed2006

Regulatory mechanisms of gene expression: complexity with elements of deterministic chaos.

Linear models based on proportionality between variables have been commonly applied in biology and medicine but in many cases they do not describe correctly the complex relationships of living organisms and now are being replaced by nonlinear theories of deterministic chaos. Recent advances in molecular biology and genome sequencing may lead to a simplistic view that all life processes in a cell, or in the whole organism, are strictly and in a linear fashion controlled by genes. In reality, the existing phenotype arises from a complex interaction of the genome and various environmental factors. Regulation of gene expression in the animal organism occurs at the level of epigenetic DNA modification, RNA transcription, mRNA translation, and many additional alterations of nascent proteins. The process of transcription is highly complicated and includes hundreds of transcription factors, enhancers and silencers, as well as various species of low molecular mass RNAs. In addition, alternative splicing or mRNA editing can generate a family of polypeptides from a single gene. Rearrangement of coding DNA sequences during somatic recombination is the source of great variability in the structure of immunoglobulins and some other proteins. The process of rearrangement of immunoglobulin genes, or such phenomena as parental imprinting of some genes, appear to occur in a random fashion. Therefore, it seems that the mechanism of genetic information flow from DNA to mature proteins does not fit the category of linear relationship based on simple reductionism or hard determinism but would be probably better described by nonlinear models, such as deterministic chaos.

Alleles↗

PubMed1992

Selective 5' modification of T7 RNA polymerase transcripts.

We have developed two methods for selective 5' modification of RNAs generated by enzymatic synthesis using T7 RNA polymerase. The first method involves a two-step procedure. Transcription reactions are performed under standard conditions except that GTP is replaced by GTP gamma S. Since the polymerase initiates transcription with GTP, every transcript contains a 5' gamma-thiophosphate group which is modified with the thiol-specific reagent of choice (e.g., iodoacetyl dansyl derivative) in the second step. In an alternative method, transcription and modification reactions are carried out in a single step, using a mixture of dansylated GTP and GTP. Under the appropriate conditions, dansylated GTP effectively competes with GTP in the initiation reaction but does not substantially inhibit the elongation reaction. Yields of fluorescent 64-mer RNA ranging from 30 to 70% of the total transcription product have been obtained using these methods in combination with HPLC purification. This approach is amenable to large scale synthesis reactions and can be used to produce a wide variety of 5'-modified RNAs of virtually any size for structural or functional studies.

Base Sequence↗

PubMedDate not supplied

[RNA synthesis and modifications of heart nuclear proteins during thyroid hormone deficiency].

RNA synthesis, correlation of various histones and acetylation and phosphorylation of the chromatin proteins were studied in the rat heart during monthly hypothyroidism. It was shown that [3H]uridine incorporation into heart RNA decreases considerably at hypothyrosis. The alteration in relative amounts of the histone H4 subfractions, which does not depend on the method of hypothyrosis reproduction (inhibition of thyroid function by 1-methyl-2-mercaptoimidazole, thyroidectomy) was detected by the method of analytical electrophoresis in 15% polyacrylamide gels containing 3.125 M urea and 0.9 N acetic acid. Increased incorporation of [32P]phosphate into histone fraction H2b and total fraction of acidic chromatin proteins was observed in vivo. Increased incorporation of labeled acetate into the total histone fraction and reduced incorporation into acidic nuclear proteins were obtained. It was shown that the increased incorporation of acetate into the total histone fraction was due to the increased acetylation of histones H3, H2b, H4 and acid-soluble chromatin proteins characteristic of tissues with a low level of replication. It is assumed that the observed changes of nuclear proteins reflect the process of chromatin reorganization caused by a prolonged deficiency of thyroid hormones.

Acetylation↗

PubMed1977

Loss of infectivity of brome mosaic virus RNA after chemical modification of the 3' or 5' terminus.

Brome mosaic virus (BMV) RNA that had both termini chemically modified by periodate oxidation and aniline-catalyzed cleavage of the terminal nucleotide had drastically reduced infectivity. BMV RNA that was first enzymatically tyrosylated to protect the 3' terminus from modification, and then modified at the 5' terminus by periodate oxidation and aniline cleavage, had a similar reduction in infectivity. Tyrosylation followed by acetylation modifies only the 3' terminus. Nevertheless, acetylated tyrosyl-BMV RNA was less than one-fourth as infectious as a control sample subjected to procedures that differed only by the presence of tyrosinol (which prevents aminoacylation and subsequent acetylation). For each modified form of viral RNA, care was taken to test the infectivity of appropriate control samples. The integrity of the modified RNAs was examined by gel electrophoresis and by biological translation and aminoacylation assays. We conclude that, in different ways, both the 5'- and 3'-terminal structures of BMV RNA play important roles during infection of the host.

Base Sequence↗

PubMedDate not supplied

Post-transcriptional modification of globin RNA.

During the last three years, significant progress has been made in the characterization of the structure of globin RNA precursors and the steps involved in their modifications to mature mRNA. Aside from the coding sequence, IVSs in RNA precursors have a critical function in the formation of mature mRNA. However, certain questions regarding the role IVSs might have in RNA processing still need to be answered. In order to understand normal globin gene expression, it will be important to know whether specific nucleoside sequences or conformational changes in the structure of the RNA due to IVSs are required for each processing enzyme in the stepwise cleavage-ligation reaction. The availability of purified RNA processing intermediates for sequencing should enable us to map the cleavage-ligation sites in each RNA. It will be interesting to determine whether any methylated nucleotides exist in IVSs and whether methylation is involved in the processing pathway. In addition, further evidence should be forthcoming to demonstrate the importance of IVS removal in the transport of RNA from nucleus to cytoplasm. Information obtained from these studies should help us to understand further the post-transcriptional control of the globin gene expression in normal states as well as in states of abnormal globin chain synthesis, such as the thalassemias.

Amino Acid Sequence↗

PubMed1997

Nuclear antisense RNA induces extensive adenosine modifications and nuclear retention of target transcripts.

Antisense RNA may regulate the expression of a number of eukaryotic genes, but little is known about its prevalence or mechanism of action. We have used a model system in which antisense control can be studied both genetically and biochemically. Late in polyoma virus infection, early-strand mRNA levels are down-regulated by nuclear antisense RNA from the late strand. Analysis of early-strand transcripts isolated late in infection revealed extensive base modifications. In many transcripts almost half of the adenosines were altered to inosines or guanosines. These results suggest modification of RNA duplexes by double-stranded RNA adenosine deaminase or a related enzyme. Probes that detect only modified RNAs revealed that these molecules are not highly unstable, but accumulate within the nucleus and are thus inert for gene expression. Antisense-induced modifications can account for most or all of the observed regulation, with the lowered levels of early-strand RNAs commonly observed late in infection resulting from the fact that many transcripts are invisible to standard hybridization probes. This work suggests that similar antisense-mediated control mechanisms may also operate under physiological conditions in uninfected eukaryotic cells, and leads to the proposal that there is a novel pool of nuclear RNAs that cannot be seen with many molecular probes heretofore used.

3T3 Cells↗

PubMed2004

The roles of histone modifications and small RNA in centromere function.

Here, epigenetic regulation of centromeric chromatin in fission yeast (Schizosaccharomyces pombe) is reviewed, focussing on the role of histone modifications and the link to RNA interference (RNAi). Fission yeast centromeres are organized into two structurally and functionally distinct domains, both of which are required for centromere function. The central core domain anchors the kinetochore structure while the flanking heterochromatin domain is important for sister centromere cohesion. The chromatin structure of both domains is regulated epigenetically. In the central core domain, the histone H3 variant Cnp1(CENP-A) plays a key role. In the flanking heterochromatin domain, histones are kept underacetylated by the histone deacetylases (HDACs) Clr3, Clr6 and Sir2, and methylated by Clr4 methyltransferase (HMTase) to create a specific binding site for the Swi6 protein. Swi6 then directly mediates cohesin binding to the centromeric heterochromatin. Recently, a surprising link was made between heterochromatin formation and RNAi.

Centromere↗

PubMed1988

Modification of ribosomal RNA by ribosome-inactivating proteins from plants.

We have surveyed 14 different toxic and nontoxic ribosome-inactivating proteins from plants for the ability to act on the RNA of the eucaryotic 60 S ribosomal subunit. All of these proteins act to introduce a specific modification into 26-28 S RNA which renders the RNA sensitive to cleavage by aniline. Sequence analysis of the 5'-termini of the fragments produced by ricin and saporin following aniline cleavage indicate that both proteins possess identical specificity. Our observations support the conclusion of Endo and Tsurugi (J. Biol. Chem. 262, 8128-8130, 1987) that ricin is a specific N-glycosidase and we have located the site of this cleavage by direct sequence analysis. Our results further suggest that all plant ribosome-inactivating proteins function as specific N-glycosidases with the same specificity.

Animals↗

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↗

PubMed1976

A comparison of the 16S ribosomal RNAs from mesophilic and thermophilic bacilli: some modifications in the Sanger method for RNA sequencing.

Two modifications in the Sanger two dimensional electrophoretic procedure for RNA analysis are reported. One increases resolution on the primary fingerprint to the point that digests of large RNAs, of the size 1500-3000 nucleotides yield well resolved fingerprint patterns. The other is a novel endonucleolytic procedure that proves useful in determining sequences of the large oligonucleotides produced by T1 ribonuclease. These modifications have been used in determining the catalogs of oligomers produced by T1 ribonuclease digestion of 16S rRNAs from three related organisms, Bacillus subtilis, B.pumilus and B.stearothermophilus. The possible effects of adaptation to a thermophilic niche on ribosomal RNA primary structure and the phylogenetic relatedness of the two mesophilic Bacilli are discussed.

Animals↗

PubMed1991

Pseudouridine modification of U5 RNA in ribonucleoprotein particles assembled in vitro.

The formation of pseudouridine (psi) in U5 RNA during ribonucleoprotein (RNP) assembly was investigated by using HeLa cell extracts. In vitro transcribed, unmodified U5 RNA assembled into an RNP particle with the same buoyant density and sedimentation velocity as did U5 small nuclear RNP from extracts. The greatest amount of psi modification was detected when a combination of S100 and nuclear extracts was used for assembly. psi formation was inhibited when ATP and creatine phosphate or MgCl2 were not included in the assembly reaction, paralleling the inhibition of RNP particle formation. A time course of assembly and psi formation showed that psi modification lags behind RNP assembly and that at very early time points, Sm-reactive U5 small nuclear RNPs are not modified. Two of three psi modifications normally found in U5 RNA were present in RNA incubated in the extracts. Mutations in the form of deletions and truncations were made in the U5 sequence, and the effect of these mutations on psi formation was investigated. A mutation in the area of stem-loop I which contains the psi moieties or in the Sm binding sequence affected psi formation.

Autoantigens↗

PubMed1994

Altered queuine modification of transfer RNA involved in the differentiation of human K562 erythroleukemia cells in the presence of distinct differentiation inducers.

Altered queuine modification of tRNA has been correlated to neoplasia and cell differentiation, but much of the existing evidence is only circumstantial. In the present study, we used several distinct differentiation inducers to measure changes in Q-family tRNA species during the erythroid differentiation of human K562 erythroleukemia cells. Treatment of K562 cells with 3.6 microM 1-beta-D-arabinofuranosylcytosine (ara-C), 1 mM sodium butyrate, 0.1 mM hemin, or 5 microM 5-azacytidine resulted in growth inhibition, erythroid differentiation, and changes in queuine content of tRNA. In the presence of the irreversible inducer ara-C, the queuine content of tRNA increased markedly when the cells differentiated into benzidine-positive erythroid cells, and cell growth was inhibited. The increase in the queuine content of tRNA in differentiated K562 cells was an irreversible event. In cells incubated with the reversible inducer sodium butyrate, an increase in the queuine content of tRNA was correlated with the increase in benzidine-positive erythroid cells throughout the culturing period. After removal of the drug at 48 h, the queuine content of tRNA decreased concomitant with a decrease in benzidine-positive cells. Treatment with another reversible inducer, hemin, caused only a transient increase in the queuine content of tRNA, which was not correlated with the steady increase in benzidine-positive erythroid cells. The agent 5-azacytidine slightly inhibited cell growth but did not significantly change the percentage of benzidine-positive cells and the queuine content of tRNA. We further clarified the changes in queuine content of tRNA by analyzing the Q-containing isoacceptors of Q-family tRNA species including tRNA(Tyr), tRNA(His), tRNA(Asp), and tRNA(Asn) by RPC-5 chromatography, and found that the change in queuine content of tRNA(Tyr) was greater than the other Q-family tRNA species during induction by ara-C, sodium butyrate, and hemin. Our results indicate that the change in queuine content of tRNA is an irreversible event of terminal differentiation in ara-C induction and is a transient event of reversible differentiation in hemin induction. Sodium butyrate induction might represent a status between irreversible and reversible differentiation. Q-containing isoacceptors of tRNA might potentially play an important biological role during K562 cell differentiation.

Aspartate-tRNA Ligase↗

PubMed1993

Ribosome activity and modification of 16S RNA are influenced by deletion of ribosomal protein S20.

A spontaneous mutant of Escherichia coli K-12 was isolated that shows an increased misreading ability of all three nonsense codons together with an inability to grow at 42 degrees C. It is demonstrated that the mutation is a deletion of the gene rpsT, coding for ribosomal protein S20. The loss of this protein not only influences the decoding properties of the ribosome; the modification pattern of 16S ribosomal RNA is also changed. This leads to a deficiency in the ability of the mutant to associate its 30S subunits with 50S subunits to form 70S ribosomes. It is suggested that two modified bases, m5C and m6(2)A, are directly or indirectly essential for association of subunits to functional ribosomes in the rpsT mutant strain. Two other modifications were also studied; m2G which is not affected at all and m3U which is undermodified in both active and inactive subunits and, therefore, not involved in subunit association.

Bacterial Proteins↗

PubMed2000

Posttranscriptional modification of transfer RNA in the submarine hyperthermophile Pyrolobus fumarii.

In the RNA of hyperthermophiles, which grow optimally between 80 degrees C and 106 degrees C, posttranscriptional modification has been identified as a leading mechanism of structural stabilization. Particularly in the Archaeal evolutionary domain these modifications are expressed as a structurally diverse array of modification motifs, many of which include ribose methylation. Using mass spectrometric techniques we have examined the posttranscriptional modifications in unfractionated tRNA from the remarkable organism Pyrolobus fumarii, which grows optimally at 106 degrees C, but up to 113 degrees C (Blöchl et al. (1997), Extremophiles, 1, 14-21). Twenty-six modified nucleosides were detected, 11 of which are methylated in ribose. A new RNA nucleoside, 1,2'-O-dimethylguanosine (m1Gm) was characterized and the structure confirmed by chemical synthesis.

Chromatography, High Pressure Liquid↗