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PubMed2006

Phosphorylation of Escherichia coli poly(A) polymerase I and effects of this modification on the enzyme activity.

In Escherichia coli, RNA polyadenylation is catalyzed mainly by poly(A) polymerase I (PAP I). Here we demonstrate that a PAP I variant with a C-terminal His tag (PAP I-His) can be phosphorylated both in vivo and in an artificial in vitro system. The in vivo phosphorylation of PAP I-His impairs activity of this enzyme. Previous studies, performed by others, indicated that phosphorylation of His-tagged proteins usually reflects such a modification of their native counterparts in bacterial cells. Therefore, our results suggest that phosphorylation and dephosphorylation of PAP I may be important regulatory processes in the control of activity of this enzyme.

Escherichia coli↗
PubMed2005

RNA editing: a molecular mechanism for the fine modulation of neuronal transmission.

The term "RNA editing" is used to identify any mechanism responsible for producing mRNA molecules with sequence information not specifically encoded in the DNA. RNA editing is therefore an important event in gene modification, which takes place at a post-transcriptional level. The molecular mechanism of RNA editing involves site-selective deamination of adenosine to inosine in pre-mRNA, which leads to altering translation codons and splicing in nuclear transcripts, whereby functionally distinct proteins can be produced from a single gene. The mammalian editing enzymes ADARs (adenosine deaminases acting on RNA) are widely expressed in brain and other tissues: however, up until now their substrates have mainly been found in the Central Nervous System (CNS). Of particular relevance in the CNS is the editing occurring at the ionotropic glutamate receptors (GluRs) level. Three AMPA and two Kainate receptors are subject to RNA editing. The consequence of this process is the substitution of specific amino acids in functionally critical positions of the receptors. Depending on the GluR involved, the consequences of editing will involve: activation and/or inhibition of splicing sites; modulation of the trafficking of the receptor to the plasma membrane; the process of tetramerization of the receptor subunits; modification of the ions passage through the receptor channel; modulation of the desensitization and action potential recovery times. All these events are specific to the different GluRs and are genetically and developmentally controlled. RNA editing is therefore a crucial event involved in controlling transmission of the action potential at the postsynaptic level. This modulation involves the transmission of all sensory stimuli to the CNS and gives rise to the "Sensotype". The Sensotype therefore defines the "way" in which the information acquired from the environment by the sensory systems is transmitted to the brain. The signals and inputs deriving from the Sensotype are transmitted to the brain, which processes and stores these signals thus generating the "Brainotype". Brainotype and Sensotype are genetically and environmentally determined; they are individually unique and specific to every living organism with a nervous system. Their characteristics are, at least in part, dependent on the modulation of the "RNA editing" process since glutamate receptors represent the main neurotransmitter system in the CNS.

Animals↗
PubMed1998

Nuclease-resistant external guide sequence-induced cleavage of target RNA by human ribonuclease P.

External guide sequences (EGSs) are short oligoribonucleotides, which are designed to bind to a given RNA target and form a precursor tRNA-like complex. This complex can be recognized by ribonuclease P (RNase P), resulting in specific cleavage of the RNA target. To explore the potential of this class of compounds as therapeutic agents and valuable tools for gene function analysis, various chemical modifications were introduced into an all-RNA EGS molecule to confer nuclease resistance. In particular, 2'-O-methyl substitutions were incorporated into the entire sequence (i.e., A-stem, D-stem, and T-stem) except the T-loop region without loss of cleavage-inducing activity. Replacement of rU (position 54) and rC (position 56) in the T-loop with their 2'-O-methyl counterparts caused pronounced decrease in activity. Moreover, phosphorothioate backbone modification of the T-loop did not provide sufficient protection against endonucleolytic attack at the ribopyrimidine residues. Systematic modification of the T-loop with a variety of modified nucleosides and the addition of a 3'-3' inverted T at the 3'-end have generated several lead EGS prototypes, which not only exhibit wild-type activity in inducing RNase P-mediated target cleavage as compared with the all-RNA control but also remain intact in human serum for more than 24 hours. These results should provide useful insights into the design and development of oligonucleotide-based EGSs as potential regulators of gene expression.

Base Sequence↗
PubMed1990

Structural analysis of a group II intron by chemical modifications and minimal energy calculations.

Folding of the yeast mitochondrial group II intron aI5c has been analysed by chemical modification of the in vitro synthesised RNA with dimethylsulfate and diethylpyrocarbonate. Computer calculations of the intron secondary structure through minimization of free energy were also performed in order to study thermodynamic properties of the intron and to relate these to data obtained from chemical modification. Comparison of the two sets of data with the current phylogenetic model structure of the intron aI5 reveals close agreement, thus lending strong support for the existence of a typical group II intron core structure comprising six neighbouring stem-loop domains. Local discrepancies between the experimental data and the model structures have been analyzed by reference to thermodynamic properties of the structure. This shows that use of the latest refined set of free energy values improves the structure calculation significantly.

Base Sequence↗
PubMed1996

Comparative analysis of tertiary structure elements in signal recognition particle RNA.

BACKGROUND: The signal recognition particle (SRP) is a ribonucleoprotein complex that associates with ribosomes to promote co-translational translocation of proteins across biological membranes. We have used comparative analysis of a large number of bacterial, archaeal, and eukaryotic SRP RNA sequences to derive shared tertiary SRP RNA structure elements. RESULTS: A representative three-dimensional model of the human SRP RNA is shown that includes single-stranded intrahelical and interhelical RNA loops and incorporates data from enzymatic and chemical modification, electron microscopy, and site-directed mutagenesis. Properties of the SRP RNA model are an overall extended dumbbell-shaped structure (260 A x 70 A) with a pseudoknot in the small SRP domain (a pairing of 12-UGGC-15 with 33-GCUA-36), and a tertiary interaction in the large SRP domain (198-GA-199 with 232-GU-233). CONCLUSIONS: The RNA 'knuckle' formed in helix 8 of SRP RNA appears to constitute the binding site for protein SRP54 or its bacterial equivalent, protein P48. A dynamic property of this feature may explain the hierarchial assembly of proteins SRP19 and SRP54 in the large SRP domain. Furthermore, the human SRP RNA model serves as a framework to understand details of the structure and function of SRP in all organisms and is presented to stimulate further experimentation in this area.

Animals↗
PubMed2002

Investigation of Overhauser effects between pseudouridine and water protons in RNA helices.

The inherent chemical properties of RNA molecules are expanded by posttranscriptional modification of specific nucleotides. Pseudouridine (psi), the most abundant of the modified bases, features an additional imino group, NH1, as compared with uridine. When psi forms a Watson-Crick base pair with adenine in an RNA helix, NH1 is positioned within the major groove. The presence of psi often increases thermal stability of the helix or loop in which it is found [Hall, K. B. & McLaughlin, L. (1992) Nucleic Acids Res. 20, 1883-1889]. X-ray crystal structures of transfer RNAs [e.g., Arnez, J. & Steitz, T. (1994) Biochemistry 33, 7560-7567] have depicted water molecules bridging psiNH1 groups and nearby phosphate oxygen atoms, but direct evidence for this interaction in solution has not been acquired. Toward this end, we have used a rotating-frame Overhauser effect spectroscopy-type NMR pulse sequence with a CLEAN chemical-exchange spectroscopy spin-lock pulse train [Hwang, T.-L., Mori, S., Shaka, A. J. & van Zijl, P. C. M. (1997) J. Am. Chem. Soc. 119, 6203-6204] to test for psiNH1-water cross-relaxation effects within two RNA helices: (i) a complementary duplex, in which psi is not associated with structural change, and (ii) an RNA duplex representing the eukaryotic pre-mRNA branch-site helix from Saccharomyces cerevisiae, in which a conserved psi extrudes the branch-site adenosine from the helix. Our data implicate a water-psiNH1 hydrogen bond both in stabilizing the complementary helix and in favoring formation of the unique structure of the branch-site helix.

Adenosine↗
PubMed2003

A second functional RNA domain in the 5' UTR of the Tomato bushy stunt virus genome: intra- and interdomain interactions mediate viral RNA replication.

The 5' untranslated regions (UTRs) of (+)-strand RNA viruses play a variety of roles in the reproductive cycles of these infectious agents. Tomato bushy stunt virus (TBSV) belongs to this class of RNA virus and is the prototype member of the genus Tombusvirus. Previous studies have demonstrated that a T-shaped domain (TSD) forms in the 5' half of the TBSV 5' UTR and that it plays a central role in viral RNA replication. Here we have extended our structure-function analysis to the 3' half of the 5' UTR. Investigation of this region in the context of a model viral replicon (i.e., a TBSV-derived defective interfering [DI] RNA) revealed that this segment contains numerous functionally relevant structural features. In vitro solution structure probing along with comparative and computer-aided RNA secondary structure analyses predicted the presence of a simple stem loop (SL5) followed by a more complex downstream domain (DSD). Both structures were found to be essential for efficient DI RNA accumulation when tested in a plant protoplast system. For SL5, maintenance of the base of its stem was the principal feature required for robust in vivo accumulation. In the DSD, both helical and unpaired regions containing conserved sequences were necessary for efficient DI RNA accumulation. Additionally, optimal DI RNA accumulation required a TSD-DSD interaction mediated by a pseudoknot. Modifications that reduced accumulation did not appreciably affect DI RNA stability in vivo, indicating that the DSD and SL5 act to facilitate viral RNA replication.

5' Untranslated Regions↗
PubMed2000

Unique structural and stabilizing roles for the individual pseudouridine residues in the 1920 region of Escherichia coli 23S rRNA.

The synthesis of a 5'-O-BzH-2'- O -ACE-protected pseudouridine phosphoramidite is reported [BzH, benzhydryloxy-bis(trimethylsilyloxy)silyl; ACE, bis(2-acetoxyethoxy)methyl]. The availability of the phosphoramidite allows for reliable and efficient syntheses of hairpin RNAs containing single or multiple pseudouridine modifications in the stem or loop regions. Five 19-nt hairpin RNAs representing the 1920-loop region (G(1906)-C(1924)) of Escherichia coli 23S rRNA were synthesized with pseudouridine residues located at positions 1911, 1915 and 1917. Thermodynamic parameters, circular dichroism spectra and NMR data are presented for all five RNAs. Overall, three different structural contexts for the pseudouridine residues were examined and compared with the unmodified RNA. Our main findings are that pseudouridine modifications exhibit a range of effects on RNA stability and structure, depending on their locations. More specifically, pseudouridines in the single-stranded loop regions of the model RNAs are slightly destabilizing, whereas a pseudo-uridine at the stem-loop junction is stabilizing. Furthermore, the observed effects on stability are approximately additive when multiple pseudouridine residues are present. The possible relationship of these results to RNA function is discussed.

Base Sequence↗
PubMed1991

RNA template-specific PCR: an improved method that dramatically reduces false positives in RT-PCR.

We report a novel modification of the reverse transcription PCR method, designated RNA template-specific PCR. With this approach, the 5' end of the first strand is tagged with a unique nucleotide sequence during reverse transcription that may then be exploited to amplify preferentially RNA-derived sequences. In our hands, RNA template-specific PCR retains the sensitivity of the traditional method, but greatly reduces the frequency of false positives, and virtually eliminates carryover contamination from PCR products amplified in previous experiments.

Animals↗
PubMed1990

N6-methyladenosine residues in an intron-specific region of prolactin pre-mRNA.

N6-methyladenosine (m6A) residues occur at internal positions in most cellular and viral RNAs; both heterogeneous nuclear RNA and mRNA are involved. This modification arises by enzymatic transfer of a methyl group from S-adenosylmethionine to the central adenosine residue in the canonical sequence G/AAC. Thus far, m6A has been mapped to specific locations in eucaryotic mRNA and viral genomic RNA. We have now examined an intron-specific sequence of a modified bovine prolactin precursor RNA for the presence of this methylated nucleotide by using both transfected-cell systems and a cell-free system capable of methylating mRNA transcripts in vitro. The results indicate the final intron-specific sequence (intron D) of a prolactin RNA molecule does indeed possess m6A residues. When mapped to specific T1 oligonucleotides, the predominant site of methylation was found to be within the consensus sequence AGm6ACU. The level of m6A at this site is nonstoichiometric; approximately 24% of the molecules are modified in vivo. Methylation was detected at markedly reduced levels at other consensus sites within the intron but not in T1 oligonucleotides which do not contain either AAC or GAC consensus sequences. In an attempt to correlate mRNA methylation with processing, stably transfected CHO cells expressing augmented levels of bovine prolactin were treated with neplanocin A, an inhibitor of methylation. Under these conditions, the relative steady-state levels of the intron-containing nuclear precursor increased four to six times that found in control cells.

Adenosine↗
PubMed2000

The tylosin resistance gene tlrB of Streptomyces fradiae encodes a methyltransferase that targets G748 in 23S rRNA.

tlrB is one of four resistance genes encoded in the operon for biosynthesis of the macrolide tylosin in antibiotic-producing strains of Streptomyces fradiae. Introduction of tlrB into Streptomyces lividans similarly confers tylosin resistance. Biochemical analysis of the rRNA from the two Streptomyces species indicates that in vivo TlrB modifies nucleotide G748 within helix 35 of 23S rRNA. Purified recombinant TlrB retains its activity and specificity in vitro and modifies G748 in 23S rRNA as well as in a 74 nucleotide RNA containing helix 35 and surrounding structures. Modification is dependent on the presence of the methyl group donor, S-adenosyl methionine. Analysis of the 74-mer RNA substrate by biochemical and mass spectrometric methods shows that TlrB adds a single methyl group to the base of G748. Homologues of TlrB in other bacteria have been revealed through database searches, indicating that TlrB is the first member to be described in a new subclass of rRNA methyltransferases that are implicated in macrolide drug resistance.

Bacterial Proteins↗
PubMed2006

Performance of the TruGene human immunodeficiency virus type 1 genotyping kit and OpenGene DNA sequencing system on clinical samples diluted to approximately 100 copies per milliliter.

The TruGene human immunodeficiency virus type 1 (HIV-1) genotyping kit/OpenGene DNA sequencing system (Bayer HealthCare, Tarrytown, NY) reliably produced clinically acceptable resistance profiles for reverse transcriptase and protease inhibitors on patient samples diluted to approximately 100 copies/ml following extraction with the QIAamp viral RNA minikit (QIAGEN Inc., Valencia, CA). One modification of the standard protocol was made to guarantee PCR amplification: a centrifugation step to concentrate virus was added before RNA extraction. For genotypic antiretroviral resistance testing, no significant differences in the identification and sensitivity of detection for codon mutations, base mutations, and multibase sites were found between the original and diluted samples.

Anti-HIV Agents↗
PubMed2004

Rational modification of a selection strategy leads to deoxyribozymes that create native 3'-5' RNA linkages.

We previously used in vitro selection to identify several classes of deoxyribozymes that mediate RNA ligation by attack of a hydroxyl group at a 5'-triphosphate. In these reactions, the nucleophilic hydroxyl group is located at an internal 2'-position of an RNA substrate, leading to 2',5'-branched RNA. To obtain deoxyribozymes that instead create linear 3'-5'-linked (native) RNA, here we strategically modified the selection approach by embedding the nascent ligation junction within an RNA:DNA duplex region. This approach should favor formation of linear rather than branched RNA because the two RNA termini are spatially constrained by Watson-Crick base pairing during the ligation reaction. Furthermore, because native 3'-5' linkages are more stable in a duplex than isomeric non-native 2'-5' linkages, this strategy is predicted to favor the formation of 3'-5' linkages. All of the new deoxyribozymes indeed create only linear 3'-5' RNA, confirming the effectiveness of the rational design. The new deoxyribozymes ligate RNA with k(obs) values up to 0.5 h(-1) at 37 degrees C and 40 mM Mg2+, pH 9.0, with up to 41% yield at 3 h incubation. They require several specific RNA nucleotides on either side of the ligation junction, which may limit their practical generality. These RNA ligase deoxyribozymes are the first that create native 3'-5' RNA linkages, which to date have been highly elusive via other selection approaches.

Base Sequence↗
PubMed1998

Modification and application of self-trimming hairpin ribozymes to targeting a transcribed RNA in vitro.

The three-domain hairpin ribozyme was improved by increasing the number of linker bases connecting domain I' with domain II from six to seven, and a new ribozyme was designed to release a trans-acting ribozyme that is able to bind with target RNAs by three hybridizing arms. The trans-cleavage activities of the trimmed ribozyme were used to cleave a long target RNA, with 85 bases transcribed from a synthetic gene encoding induced nitric oxide synthase (iNOS). Although the ribozyme with three-hybridizing arms did not efficiently cleave the long target RNA, the overall activity, from self-trimming to the trans-cleavage reaction of the target RNA, was higher than that of the ribozyme with two hybridizing arms.

Base Sequence↗
PubMed2004

The RNA binding protein Sam68 is acetylated in tumor cell lines, and its acetylation correlates with enhanced RNA binding activity.

Sam68 (Src-associated in mitosis; 68 kDa) is a member of the STAR (signal transduction and activation of RNA) family of KH domain-containing RNA binding proteins. Accumulating evidence suggests that it plays an important role in cell cycle control. Tyrosine phosphorylation by Src family kinases and breast tumor kinase can negatively regulate its RNA binding activity. To date, there are no reports of a factor, such as a phosphatase, which can positively regulate Sam68 association with RNA. Acetylation is a reversible post-translational modification known to influence the activity of DNA binding proteins. However, acetylation of a cellular RNA binding protein as a mechanism for regulating its activity has not yet been reported. Here we demonstrate Sam68 to be acetylated in vivo. A screen of several human mammary epithelial cell lines revealed variations in Sam68 acetylation. Interestingly, the highest level of acetylation was found in tumorigenic breast cancer cell lines. The screen also showed a positive correlation between Sam68 acetylation and its ability to bind RNA. The acetyltransferase CBP was shown to acetylate Sam68 and enhance its binding to poly(U) RNA. These results suggest that Sam68 association with RNA substrates may be positively regulated by acetylation, and that enhanced acetylation and RNA binding activity of Sam68 may play a role in tumor cell proliferation.

Acetylation↗
PubMed1998

Disassociation of sigma subunit from RNA polymerase of Xanthomonas oryzae pv. oryzae by phage Xp10 infection.

The sigma subunit of Xanthomonas oryzae pv. oryzae is disassociated from host RNA polymerase after phage Xp10 infection. To clarify the possible mechanism for this observation, sigma subunit was purified and an antiserum against sigma subunit was prepared. Immunoprecipitation of RNA polymerase by the anti-core RNA polymerase antiserum, followed by immunoblotting with anti-sigma subunit antibody, revealed that sigma subunit was lost from RNA polymerase within 10 minutes after Xp10 infection. Loss of sigma subunit was not observed under other stress conditions including heat and cold stress, starvation and growth to stationary phase. Two-dimensional immunoblotting analysis did not reveal any covalent modification of either sigma subunit or RNA polymerase after Xp10 infection. These results suggest that separation of th subunit from RNA polymerase may be due to competition with other binding factors.

Antibodies, Bacterial↗
PubMed1966

Effect of thymine starvation on messenger ribonucleic acid synthesis in Escherichia coli.

Luzzati, Denise (Institut de Biologie Physico-Chimique, Paris, France). Effect of thymine starvation on messenger ribonucleic acid synthesis in Escherichia coli. J. Bacteriol. 92:1435-1446. 1966.-During the course of thymine starvation, the rate of synthesis of messenger ribonucleic acid (mRNA, the rapidly labeled fraction of the RNA which decays in the presence of dinitrophenol or which hybridizes with deoxyribonucleic acid) decreases exponentially, in parallel with the viability of the thymine-starved bacteria. The ability of cell-free extracts of starved bacteria to incorporate ribonucleoside triphosphates into RNA was determined; it was found to be inferior to that of extracts from control cells. The analysis of the properties of cell-free extracts of starved cells shows that their decreased RNA polymerase activity is the consequence of a modification of their deoxyribonucleic acid, the ability of which to serve as a template for RNA polymerase decreases during starvation.

Carbon Isotopes↗