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At least 325 records · Page 18Linked to original sources
PubMed1998

A novel acidophilic RNA motif that recognizes coenzyme A.

Specific recognition of nucleotide cofactors by RNA may be important in engineering new RNA enzymes (ribozymes). Although in vitro selections (SELEX) have identified nucleic acid motifs ("aptamers") that bind a variety of adenosine cofactors, none of these recognizes coenzyme A (CoA), the primary biological cofactor used in acyltransfer reactions. We used SELEX experiments with two random RNA pools to identify aptamers that bind CoA. Functional boundary determination and extensive comparative sequence analysis (including reselection of a mutagenized, circularly permuted RNA) led to the identification of a 52 nucleotide minimal aptamer ("min52"). The RNA structural motif contains a large internal loop with 26 unpaired nucleotides flanked by helices of any base-paired sequence. Twenty loop nucleotides are specifically required for binding activity, 12 of which are derived from the original primer binding sequences. Specificity studies with CoA analogues demonstrated that the aptamer recognizes many adenosine analogues, including ATP, and that recognition is predominantly through the Höogsteen face of adenine. Binding activity is greatest at acidic pH (optimum near 5.0), in low or no monovalent salt, and at high concentrations of either Mg2+ or Mn2+. Strong binding activity (86% of maximum) is observed at pH 4.0, suggesting that at least some extreme conditions (acidic pH) may be compatible with RNA World theories of the origin and early evolution of life. In the presence of 10 mM Mg2+, binding is unaffected by the addition of 1 mM Ca2+, but it is mildly inhibited by 1 mM Zn2+ or Co2+ or by 0.1 mM Cu2+ or Ni2+. The dissociation constant (Kd) for the association of min52 RNA with ATP in solution was measured to be 2.4 +/- 0.4 microM under the conditions of the selection and 0.5 +/- 0.1 microM under optimized conditions. Finally, we show that the selected CoA aptamer populations contain other RNAs at low frequencies that preferentially recognize intact CoA and are not eluted from the resin by AMP alone.

Adenosine↗
PubMed1979

Evidence for a partial RNA transcript of the small circular component of kinetoplast DNA of Crithidia acanthocephali.

The major component of kinetoplast DNA (kDNA) in the protozoan Crithidia acanthocephali is an association of approximately 27,000, 0.8 micrometers (1.58 x 10(6) dalton) circular molecules apparently held together in a particular structural configuration by topological interlocking. We have carried out hybridization experiments between kDNA samples containing one or the other of the two complementary (H and L) strands of purified 0.8 micrometers molecules derived from mechanically disrupted associations and RNA samples prepared either from whole C. acanthocephali cells or from a mitochondrion-enriched fraction. The results of experiments involving cesium sulfate buoyant density centrifugation indicate that whole cell RNA contains a component(s) complementary to all kDNA H strands, but none complementary to kDNA L strands. Similar results were obtained using mitochondrion-associated RNA. Digestion of RNA/DNA hybrids and suitable controls with the single-strand-specific nuclease S1 indicated that 10% of the kDNA H strand is involved in hybrid formation. Visualization of RNA/DNA hybrids stained with bacteriophage T4 gene 32 protein revealed that hybridation involves a single region of each kDNA H strand, equal to approximately 10% of the molecule length. These data suggest that at least 10% of the small circular component of kDNA of Crithidia acanthocephali is transcribed.

Animals↗
PubMed2006

Structural stabilization of GTP-binding domains in circularly permuted GTPases: implications for RNA binding.

GTP hydrolysis by GTPases requires crucial residues embedded in a conserved G-domain as sequence motifs G1-G5. However, in some of the recently identified GTPases, the motif order is circularly permuted. All possible circular permutations were identified after artificially permuting the classical GTPases and subjecting them to profile Hidden Markov Model searches. This revealed G4-G5-G1-G2-G3 as the only possible circular permutation that can exist in nature. It was also possible to recognize a structural rationale for the absence of other permutations, which either destabilize the invariant GTPase fold or disrupt regions that provide critical residues for GTP binding and hydrolysis, such as Switch-I and Switch-II. The circular permutation relocates Switch-II to the C-terminus and leaves it unfastened, thus affecting GTP binding and hydrolysis. Stabilizing this region would require the presence of an additional domain following Switch-II. Circularly permuted GTPases (cpGTPases) conform to such a requirement and always possess an 'anchoring' C-terminal domain. There are four sub-families of cpGTPases, of which three possess an additional domain N-terminal to the G-domain. The biochemical function of these domains, based on available experimental reports and domain recognition analysis carried out here, are suggestive of RNA binding. The features that dictate RNA binding are unique to each subfamily. It is possible that RNA-binding modulates GTP binding or vice versa. In addition, phylogenetic analysis indicates a closer evolutionary relationship between cpGTPases and a set of universally conserved bacterial GTPases that bind the ribosome. It appears that cpGTPases are RNA-binding proteins possessing a means to relate GTP binding to RNA binding.

Amino Acid Sequence↗
PubMed2006

Assembly of the archaeal box C/D sRNP can occur via alternative pathways and requires temperature-facilitated sRNA remodeling.

Archaeal dual-guide box C/D small nucleolar RNA-like RNAs (sRNAs) bind three core proteins in sequential order at both terminal box C/D and internal C'/D' motifs to assemble two ribonuclear protein (RNP) complexes active in guiding nucleotide methylation. Experiments have investigated the process of box C/D sRNP assembly and the resultant changes in sRNA structure or "remodeling" as a consequence of sRNP core protein binding. Hierarchical assembly of the Methanocaldococcus jannaschii sR8 box C/D sRNP is a temperature-dependent process with binding of L7 and Nop56/58 core proteins to the sRNA requiring elevated temperature to facilitate necessary RNA structural dynamics. Circular dichroism (CD) spectroscopy and RNA thermal denaturation revealed an increased order and stability of sRNA folded structure as a result of L7 binding. Subsequent binding of the Nop56/58 and fibrillarin core proteins to the L7-sRNA complex further remodeled sRNA structure. Assessment of sR8 guide region accessibility using complementary RNA oligonucleotide probes revealed significant changes in guide region structure during sRNP assembly. A second dual-guide box C/D sRNA from M. jannaschii, sR6, also exhibited RNA remodeling during temperature-dependent sRNP assembly, although core protein binding was affected by sR6's distinct folded structure. Interestingly, the sR6 sRNP followed an alternative assembly pathway, with both guide regions being continuously exposed during sRNP assembly. Further experiments using sR8 mutants possessing alternative guide regions demonstrated that sRNA folded structure induced by specific guide sequences impacted the sRNP assembly pathway. Nevertheless, assembled sRNPs were active for sRNA-guided methylation independent of the pathway followed. Thus, RNA remodeling appears to be a common and requisite feature of archaeal dual-guide box C/D sRNP assembly and formation of the mature sRNP can follow different assembly pathways in generating catalytically active complexes.

Amino Acid Sequence↗
PubMed1995

Identification of a retroviroid-like element from plants.

The biological nature of carnation small viroid-like RNA (CarSV RNA), a 275-nt circular molecule with self-cleaving hammerhead structures in its strands of both polarities, was investigated. The lack of infectivity observed in a series of transmission assays in carnation indicates that CarSV RNA, in spite of sharing structural similarities with viroid and viroid-like satellite RNAs from plants, does not belong to either of these two groups. Additional evidence in this direction comes from the observation that CarSV RNA also exists in carnation plants as DNA tandem repeats. In this respect, CarSV RNA is similar to a small transcript of a tandemly repeated DNA sequence of the newt genome. Moreover, CarSV and newt RNAs have similarities in their sequences as well as in some characteristics of their corresponding hammerhead structures. Further analyses have revealed that CarSV DNA is found directly fused to DNA sequences of carnation etched ring caulimovirus, a pararetrovirus, most likely in the form of an extrachromosomal element. The properties of the CarSV RNA/DNA system are those of a retroviroid-like element having some features in common with viroid and viroid-like satellite RNAs from plants and others with the newt transcript.

Base Sequence↗
PubMed1978

Glutathione-facilitated refolding of reduced, denatured bovine seminal ribonuclease: kinetics and characterization of products.

Totally reduced and denatured seminal ribonuclease was regenerated using the glutathione redox system. The refolding kinetics of the enzyme were determined as a function of redox state, temperature from 14 to 43 degrees C, pH, and protein concentration. The maximal rate of regeneration occurred with 3 x 10(-3) M reduced glutathione, 6 x 10(-4) M oxidized glutathione, 24 to 30 degrees C, and pH 8.2. The products of the refolding process were characterized by Sephadex G-75, sodium dodecyl sulfate gel electrophoresis, enzymatic activity, circular dichroism, and amino acid analysis. The results indicate that the native dimeric form of the enzyme is not produced during refolding to any appreciable extent; rather, the major product is monomeric. The purified monomer exhibits twice the activity of the native enzyme toward yeast RNA. Its circular dichroism spectrum is different from the native enzyme and is quite similar to that of pancreatic ribonuclease A. Amino acid analyses showed that two glutathione molecules are bound to the monomer, suggesting that cysteine-31 and -32, which normally form the intermolecular disulfide bonds, are blocked.

Amino Acids↗
PubMed1996

Pre-mRNA topology is important for 3'-end formation in Saccharomyces cerevisiae and mammals.

Various signal motifs that are required for efficient pre-mRNA 3'-end formation in the yeast Saccharomyces cerevisiae have been reported. None of these known signal sequences appears to be of the same general importance as is the mammalian AAUAAA motif. To establish the importance of yeast pre-mRNA termini in 3'-end formation, the ends of a pre-mRNA transcript synthesized in vitro were ligated before incubation in a yeast whole-cell extract. Such covalently closed circular RNAs were not cleaved at their poly(A) sites. Interestingly, pseudocircular RNAs with complementary 3'- and 5'-terminal sequences allowing the formation of panhandle structures were also resistant to cleavage. However, 3'-end processing was impeded neither by terminal hairpins at either or at both ends nor by RNA oligonucleotides complementary to either or both ends of a linear pre-mRNA. Intriguingly mammalian pseudocircular pre-mRNAs also were not cleaved at their poly(A) sites when incubated in a HeLa cell nuclear extract. These results provide evidence for the general importance of RNA topology in the formation of an active 3'-end processing complex in S. cerevisiae and higher eukaryotes. The possibility of a torus-shaped factor involved in 3'-end formation is discussed.

Alcohol Dehydrogenase↗
PubMed1985

Molecular cloning of potato spindle tuber viroid (PSTV) cDNA synthesized by enzymatic elongation of PSTV-specific DNA primers: a general strategy for viroid cloning.

Different cDNAs were synthesized by primer extension from the RNA of the severe strain KF 440 of potato spindle tuber viroid (PSTV) with the aid of reverse transcriptase using three PSTV-specific DNA molecules as primers. The cDNAs were made double-stranded and cloned into plasmid pBR 322. Various overlapping subgenomic DNA fragments were prepared from these clones and recombined in two different ways. In both cases a PSTV DNA copy was obtained which represented the entire PSTV RNA genome. The sequence of the DNA of one of the resulting full-length clones was identical with the original PSTV isolate, whereas the other clone showed one nucleotide change. On the basis of these results the advantages and problems of different strategies for the molecular cloning of the circular viroid RNA genome are discussed.

Base Sequence↗
PubMed1988

Purification and characterization of an endonuclease (E.C. 3.1.30.1) from Streptomyces tendae.

A single-strand-specific endonuclease which converted negatively supercoiled DNA to open-circular and linear DNA was purified to homogeneity with Hb-Sepharose 4B, DEAE Trisacryl M, HA-Ultrogel and PBE-94 chromatofocusing from extracts of Streptomyces tendae ATCC 31160. Bio-Gel P-200 chromatography and electrophoresis in SDS-PAGE indicated the native protein was a monomer with a molecular weight of approximately 40-kDa. This enzyme did not hydrolyze double-stranded linear DNA but digested RNA and circular single-strand DNA. Sequence specificity for nicking of negatively supercoiled DNA was not detected.

Chemical Fractionation↗
PubMed1988

Determination of the RNA-content in samples of isolated chromatin by circular dichroism measurements.

A method is presented to determine the RNA content in isolated chromatin. An increased ellipticity at 260 nm-290 nm in the circular dichroism spectrum of chromatin is due to RNA released together with chromatin from the cell nuclei. Mixtures of chicken erythrocyte chromatin and pure 5s-RNA were used to reconstruct such spectra. From the obtained data an RNA content higher than 3-5% in samples of isolated chromatin can be determined.

Animals↗
PubMed1998

An Arg/Lys-rich core peptide mimics TRBP binding to the HIV-1 TAR RNA upper-stem/loop.

TRBP is a cellular protein that binds to the HIV-1 leader RNA, TAR. Circular dichroism experiments have shown that a 24 amino acid peptide (TR1), located within a dsRNA binding domain (dsRBD) of TRBP, binds TAR with a 3:1 stoichiometry, eliciting a conformational change involving base unstacking. The binding characteristics of synthetic structural variants of TAR indicate that guanine residues play a key role in the TR1-RNA interaction and that binding sites exist in the upper-stem/loop and lower stem region of TAR. Deletion analysis of TR1 has led to the identification of a 15 amino acid subpeptide (TR13) which is necessary and sufficient to bind to the high affinity upper-stem/loop binding site of TAR. Alanine scanning of TR13 has revealed that mutations in either Lys or Arg residues result in altered TAR-binding, and molecular modelling/docking experiments have shown that the two Arg residues of TR13 can interact with two appropriately spaced guanine residues in the upper-stem/loop of TAR. The TR13 lysine residues appear to be essential for maintaining structural integrity and the correct positioning of the Arg side-chains. We propose that TRBP binds TAR by means of a "2-G hook" motif and that the binding specificity of this particular member of the family of double-stranded RNA-binding proteins lies within the highly conserved dsRBD core motif. Finally, our results also suggest that TRBP may function in vivo by modifying the tertiary structure of TAR RNA.

Amino Acid Sequence↗
PubMed1985

Reactions of the intervening sequence of the Tetrahymena ribosomal ribonucleic acid precursor: pH dependence of cyclization and site-specific hydrolysis.

During self-splicing of the Tetrahymena rRNA precursor, the intervening sequence (IVS) is excised as a unique linear molecule and subsequently cyclized. Cyclization involves formation of a phosphodiester bond between the 3' end and nucleotide 16 of the linear RNA, with release of an oligonucleotide containing the first 15 nucleotides. We find that the rate of cyclization is independent of pH in the range 4.7-9.0. A minor site of cyclization at nucleotide 20 is characterized. Cyclization to this site becomes more prominent at higher pHs, although under all conditions examined it is minor compared to cyclization at nucleotide 16. The circular IVS RNAs are unstable, undergoing hydrolysis at the phosphodiester bond that was formed during cyclization. We find that the rate of site-specific hydrolysis is first order with respect to hydroxide ion concentration, with a rate constant 10(3)-10(4)-fold greater than that of hydrolysis of strained cyclic phosphate esters. On the basis of these results, we propose that circular IVS RNA hydrolysis involves direct attack of OH- on the phosphate at the ligation junction, that particular phosphate being made particularly reactive by the folding of the RNA molecule. Cyclization, on the other hand, appears to occur by direct attack of the 3'-terminal hydroxyl group of the linear IVS RNA without prior deprotonation.

Animals↗
PubMed2026

A novel peptide encoded by circTLL1 drives osimertinib resistance in lung cancer by modulating the NT5C2/Ras/PI3K axis.

BACKGROUND: Acquired resistance to osimertinib, a third-generation EGFR tyrosine kinase inhibitor, remains a major clinical challenge in the treatment of non-small cell lung cancer (NSCLC). Although circular RNAs (circRNAs) have been increasingly implicated in drug resistance, most studies have focused on their canonical role as microRNA sponges, while their capacity to encode functional micropeptides remains largely unexplored. This study aimed to identify novel circRNAs involved in osimertinib resistance and to characterize their regulatory functions at the protein level. METHODS: Osimertinib-resistant (OR) NSCLC cell lines were established and validated. High-throughput RNA sequencing was performed to compare the circRNA expression profiles between parental and OR cells. The function of the candidate circRNA was assessed through a series of in vitro and in vivo experiments, including cell viability assays, apoptosis analysis, and xenograft mouse models. Mechanistic investigations involved mass spectrometry, co-immunoprecipitation and western blotting to explore its protein-coding potential and downstream signaling pathways. RESULTS: We identified a novel circRNA, termed circTLL1, that was stably and significantly upregulated in OR-NSCLC cells. Functionally, overexpression of circTLL1 promoted osimertinib resistance, whereas its knockdown restored drug sensitivity both in vitro and in vivo. Mechanistically, we discovered that circTLL1 harbors an open reading frame (ORF) that is translated into a novel 90-amino-acid protein, which we designated circTLL1-90aa. Further investigation revealed that circTLL1-90aa directly interacts with and promotes the degradation of 5'-nucleotidase, cytosolic II (NT5C2), thereby uncoupling nucleotide metabolism from its normal regulatory constraints. The consequent downregulation of NT5C2 leads to elevated GTP levels and leading to the sustained activation of the downstream Ras/PI3K/AKT signaling pathway. CONCLUSION: Our findings unveil a previously unrecognized circRNA/micropeptide/metabolism cascade underlying osimertinib resistance. The identification of the circTLL1-90aa/NT5C2/Ras/PI3K axis not only expands the functional repertoire of the non-coding genome but also provides new insights into the complexity of drug resistance. Given its selective upregulation in resistant cells, circTLL1-90aa holds promise both as a predictive biomarker for treatment stratification and as an actionable therapeutic target, offering a novel strategy to overcome osimertinib resistance in NSCLC patients.

Pyrimidines↗
PubMed1992

Processing of linear longer-than-unit-length potato spindle tuber viroid RNAs into infectious monomeric circular molecules by a G-specific endoribonuclease.

Different cDNA constructs were used for the in vitro synthesis of RNA transcripts that contain a complete monomeric unit of the potato spindle tuber viroid (PSTVd) plus an additional repeat of a part of the circular RNA genome. These permutated linear longer-than-unit-length PSTVd RNAs were incubated with the G-specific endoribonuclease RNase T1 which generated monomeric circular PSTVd RNA molecules that were infectious when mechanically inoculated to tomato plants. Besides the correct monomeric PSTVd RNA, smaller and larger circular RNAs were also formed during the reaction. The comparison of different transcripts revealed that correct in vitro processing of PSTVd RNA can proceed at alternative sites indicating that circularization is driven by RNA structure and not governed by a particular sequence. Based on these data, we propose a novel model for the processing of multimeric replicative viroid RNA intermediates through RNA cleavage and ligation catalyzed by a host endoribonuclease.

Base Sequence↗
PubMed1996

A single nucleotide substitution converts potato spindle tuber viroid (PSTVd) from a noninfectious to an infectious RNA for nicotiana tabacum.

Mechanical inoculation of Nicotiana tabacum with the PSTVd isolate KF 440-2 from the host plant tomato resulted in the de novo emergence, replication, and accumulation of a new "tobacco variant," designated PSTVd NT. It produces no symptoms in tobacco but, like PSTV KF 440-2, severe ones in tomato. The sequence analysis of PSTVd NT revealed a single nucleotide substitution from C-->U at position 259. Autonomous viroid replication was also induced in tobacco by genomic integration of oligomeric cDNA copies of PSTVd KF 440-2. Although these cDNAs contained the original tomato-specific C259, the circular PSTVd RNA subsequently accumulating in tobacco also exhibited the C259-->U259 substitution. In the secondary structure of PSTVd, nucleotide 259 is part of an internal loop analogous to loop E of eukaryotic 5S rRNA and presumed to be the only bulged extrahelical nucleotide of this loop. The C259 in PSTVd KF 440-2 and in practically all other isolates and the U259 in PSTVd NT of the loop E-like structure might be involved in protein binding and in viroid processing. The new variant PSTVd NT is genetically stable in both tobacco and tomato.

Base Composition↗
PubMed2002

Complete genome sequence, taxonomic assignment, and comparative analysis of the untranslated regions of the Modoc virus, a flavivirus with no known vector.

We recently developed a model for flavivirus infection in mice and hamsters using the Modoc virus (MODV), a flavivirus with no known vector (P. Leyssen, A. Van Lommel, C. Drosten, H. Schmitz, E. De Clercq, and J. Neyts, 2001, Virology 279, 27-37). We now present the coding and noncoding sequence of MODV. The Modoc virus genome was determined to be 10,600 nucleotides in length with a single open reading frame extending from nucleotides 110 to 10,234, encoding 3374 amino acids. The deduced gene order of the single open reading frame is C-prM-E-NS1-NS2A-NS2B-NS3-NS4A-NS4B-NS5, which is exactly the same as that of the mosquito- and tick-borne flaviviruses. It is flanked by a 5'- and 3'-untranslated region (UTR) of 109 and 366 nucleotides, respectively. Alignment of the MODV amino acid sequence with that of 20 other flaviviruses revealed several regions with high sequence similarity corresponding to functionally important domains (e.g., the serine protease/helicase/NTPase of NS3 and the methyltransferase/RNA-dependent RNA polymerase of NS5) and conserved sites for proteolytic cleavage by viral and cellular proteases. Phylogenetic analysis of the entire coding region confirmed the classification of MODV within the flaviviruses with no known vector, which is in agreement with previous findings based on partial NS5 sequences. A detailed comparative analysis of the putative folding patterns of the 5'- and 3'-UTR of MODV and of the tick- and mosquito-borne viruses was carried out. Structural elements in the 5'- and 3' UTR of MODV that are preserved among vector-borne flaviviruses were noted and so were structural elements distinguishing the MODV UTRs from mosquito-borne and tick-borne flaviviruses. Also the putative secondary structure of circularized MODV RNA is presented.

3' Untranslated Regions↗
PubMed1983

Mitochondrial circular RNAs are absent in sporulating cells of Saccharomyces cerevisiae.

During sporulation of Saccharomyces cerevisiae the mitochondria undergo a differentiation process, leading to a "spore mitochondrion", which differs both physiologically and morphologically from vegetative cell mitochondria. We report here the behaviour of the mitochondrial transcripts during this differentiation process. The circular transcripts representing spliced introns of the two mitochondrial split genes for cytochrome b and for the subunit 1 of the cytochrome c oxidase, which are very abundant during vegetative growth, are not detectable in sporulating cells. This loss does neither take place in haploid cells under sporulation conditions nor when erythromycin is added to the medium. The process of some mRNAs is advanced in relation to their precursors in sporulating cells.

Base Sequence↗