Multiple exon skipping and RNA circularisation contribute to the severe phenotypic expression of exon 5 dystrophin deletion.
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Alternative Splicing↗
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A microRNA detection system based on padlock probes and rolling circle amplification.
The differential expression and the regulatory roles of microRNAs (miRNAs) are being studied intensively these years. Their minute size of only 19-24 nucleotides and strong sequence similarity among related species call for enhanced methods for reliable detection and quantification. Moreover, miRNA expression is generally restricted to a limited number of specific cells within an organism and therefore requires highly sensitive detection methods. Here we present a simple and reliable miRNA detection protocol based on padlock probes and rolling circle amplification. It can be performed without specialized equipment and is capable of measuring the content of specific miRNAs in a few nanograms of total RNA.
Animals↗
A general two-step strategy to synthesize lariat RNAs.
We describe a general and efficient two-step strategy for lariat RNA synthesis. In the first step, a deoxyribozyme synthesizes 2',5'-branched RNA. In the second step, T4 RNA ligase closes the loop that completes the lariat. The loop-closure reaction can form either a natural or unnatural lariat isomer, depending on which of the two 3'-termini of the branched RNA reacts with the lone 5'-end. We demonstrate two approaches to control formation of either lariat isomer. In conjunction with other routes for lariat RNA synthesis, the two-step strategy described here will facilitate biochemical studies that require lariat RNAs of varying nucleotide sequence.
Base Sequence↗
The enzymatic conversion of 3'-phosphate terminated RNA chains to 2',3'-cyclic phosphate derivatives.
The enzyme, RNA cyclase, has been purified from cell-free extracts of HeLa cells approximately 6000-fold. The enzyme catalyzes the conversion of 3'-phosphate ends of RNA chains to the 2',3'-cyclic phosphate derivative in the presence of ATP or adenosine 5'-(gamma-thio)triphosphate (ATP gamma S) and Mg2+. The formation of 1 mol of 2',3'-cyclic phosphate ends is associated with the disappearance of 1 mol of 3'-phosphate termini and the hydrolysis of 1 mol of ATP gamma S to AMP and thiopyrophosphate. No other nucleotides could substitute for ATP or ATP gamma S in the reaction. The reaction catalyzed by RNA cyclase was not reversible and exchange reactions between [32P]pyrophosphate and ATP were not detected. However, an enzyme-AMP intermediate could be identified that was hydrolyzed by the addition of inorganic pyrophosphate or 3'-phosphate terminated RNA chains but not by 3'-OH terminated chains or inorganic phosphate. 3'-[32P](Up)10Gp* could be converted to a form that yielded, (Formula: see text) after degradation with nuclease P1, by the addition of wheat germ RNA ligase, 5'-hydroxylpolynucleotide kinase, RNA cyclase, and ATP. This indicates that the RNA cyclase had catalyzed the formation of the 2',3'-cyclic phosphate derivative, the kinase had phosphorylated the 5'-hydroxyl end of the RNA, and the wheat germ RNA ligase had catalyzed the formation of a 3',5'-phosphodiester linkage concomitant with the conversion of the 2',3'-cyclic end to a 2'-phosphate terminated residue.
Adenosine Triphosphate↗
Circular dichroism studies suggest that TAR RNA changes conformation upon specific binding of arginine or guanidine.
Short basic peptides from the HIV Tat protein bind specifically to a bulge region in TAR RNA, with a single arginine residue providing the only sequence-specific contact. The free amino acid arginine also binds specifically to TAR. Previous circular dichroism (CD) experiments suggested that peptide binding induces a conformational change in TAR. Here we confirm this observation using single arginine-containing peptides and show that arginine or guanidine binding also induces a conformational change in TAR. A peptide containing a single arginine within a stretch of histidines (CYHHHRHHHHHA) shows pH-dependent binding and a corresponding change in TAR conformation, as detected by a decrease in the CD signal at 265 nm. Arginine and guanidine, which bind to TAR with apparent Kd's of approximately 1.5 mM, induce similar CD changes. In contrast, lysine, which does not bind specifically to TAR, has no effect. Mutants of TAR that abolish specific binding (a U-->C substitution in the three-nucleotide bulge, a deletion of the bulge, or an A-U to U-A base pair change above the bulge) show no change in the CD signal upon binding of peptides, arginine, or guanidine. The results suggest that binding of a single guanidinium group to a specific site in TAR induces a change in RNA conformation.
Amino Acid Sequence↗
Use of circular permutation and end modification to position photoaffinity probes for analysis of RNA structure.
Photocrosslinking allows first-order structural analysis with relatively small amounts of biological material and can be applied in complex in vitro systems. In this article we describe methods for positioning both arylazide and thionucleotide photoagents within an RNA of interest by end modification of circularly permuted RNAs. Application of this technique provided a library of constraints that, together with biochemical and phylogenetic comparative data, were used to develop a structure model of the bacterial ribonuclease P ribozyme-substrate complex. Circularly permuted genes for in vitro transcription are generated by PCR from tandem genes. Circularly permuted RNA transcripts can be modified with high efficiency at both the 5' and 3' termini with arylazide crosslinking reagents, or transcription can be primed with photoactive nucleotide analog monophosphates such as 6-thioguanosine. These crosslinking agents can be used over a wide range of experimental conditions but remain inert until they are activated by UV light. Crosslinked sites are subsequently mapped by reverse transcriptase primer extension of gel-purified crosslinked species. In addition to providing basic protocols for these methods, we discuss approaches for establishing the relevance of crosslinking data to native RNA structure.
Aminophylline↗
Circular dichroism melting studies on R17 phage RNA.
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Circular Dichroism↗
Telomerase recognizes its template by using an adjacent RNA motif.
Telomerase adds telomeric repeats to chromosome 3' ends, forestalling the cellular senescence, apoptosis, and genomic instability that result from telomere loss caused by incomplete DNA replication. The telomerase ribonucleoprotein is dedicated to synthesis of tandem, simple-sequence repeats by virtue of its specialization for copying only a specific template region within the integral RNA. Here, using circularly permuted variants of Tetrahymena thermophila telomerase RNA, we identify the features that allow recognition of the template region within the RNA. We engineered a template-less telomerase ribonucleoprotein that can position and reverse transcribe an exchangeable RNA oligonucleotide template accurately. Only a short "template-recognition" element sequence tag is required to direct efficient use of adjacent 5' residues as a template for telomeric repeat synthesis. Our findings reveal molecular requirements for template selection by telomerase and physically resolve templating from other RNA functions in catalysis.
Animals↗
Circular dichroism of double-stranded RNA in the presence of salt and ethanol.
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Circular Dichroism↗
Enhanced resolution of circular and linear molecular forms of viroid and viroid-like RNA by electrophoresis in a discontinuous-pH system.
A discontinuous-pH polyacrylamide gel electrophoresis system is described. An increase in the pH differential between the gel and the running buffer enhances the separation of low molecular weight circular and linear RNA molecules. Highly purified preparations of the circular form of viroids can be obtained with this procedure. Since all the linear RNAs of similar molecular weight migrate with the front, a relatively clean background can be obtained even when crude extracts are used. This facilitates an improved separation and identification of similarly sized viroid-like RNAs. The conditions of electrophoresis in low salt and 8 M urea also permit the effective transfer of RNA molecules directly to nylon-based membranes without any additional denaturation treatment.
Electrophoresis, Polyacrylamide Gel↗
A circular DNA in malaria parasites encodes an RNA polymerase like that of prokaryotes and chloroplasts.
A 3.5-kb Sau3AI fragment was cloned from a circular DNA molecule isolated from the human malaria parasite Plasmodium falciparum and found to contain two contiguous open reading frames. These encode portions of beta and beta' subunits of an RNA polymerase similar to prokaryotic and chloroplast RNA polymerases, and contain highly conserved structural elements. The Plasmodium genes are arranged in a polycistronic transcription unit, as in both Escherichia coli and chloroplast genomes, and are transcribed in erythrocytic stages. These results suggest that the circular DNA may be an unusual mitochondrial DNA, or derived from an unidentified organelle. Because the beta subunit of prokaryotic RNA polymerases is the specific target of the antibiotic rifampicin, our observations may explain the high sensitivity of P. falciparum to this drug in vitro and indicate a new target for chemotherapy.
Amino Acid Sequence↗
Entamoeba histolytica ribosomal RNA genes are carried on palindromic circular DNA molecules.
Highly abundant DNA fragments obtained after restriction enzyme digests of nuclear DNA of Entamoeba histolytica strain HM-1:IMSS have been cloned and characterized. Northern blot hybridization to E. histolytica rRNA and sequence analysis identified the abundant DNAs as ribosomal DNA containing species. Several overlapping clones containing these abundant DNAs were isolated from 4 different genomic libraries of E. histolytica. Alignment of the restriction maps was consistent with a circular molecule, about 24.6 kilobase pairs (kb) in size. Nuclease BA131 digestion provided additional evidence for the circular nature of this DNA. The ribosomal DNA molecule contains two large inverted repeat-regions, each at least 5.2 kb in length. Sequence analysis of clone R715 revealed homology to the large rRNA units of various eukaryotic organisms. This clone was located in both inverted repeats, suggesting two rRNA cistrons per molecule. The inverted repeats are flanked by stretches of DNA which contain tandemly reiterated sequences. Southern blot analysis of E. histolytica nuclear DNA revealed the presence of two populations of molecules. These molecules have identical arrangements of restriction sites, but differ in size (0.7 kb) in a fragment containing tandemly reiterated sequences. Analysis of E. histolytica nuclear DNA by electron microscopy also revealed circular molecules. These molecules are about 26.6 kb +/- 0.5 kb in size and contain structural features predicted by the restriction map of the extrachromosomal ribosomal DNA of E. histolytica.
Animals↗
Plant pathogenic RNAs and RNA catalysis.
The rolling circle replication of small circular plant pathogenic RNAs requires a processing step to convert multimeric intermediates to monomers which are then circularized. Eleven such RNAs are known so far, two are viroids, one is viroid-like and the remainder are satellite RNAs dependent on a helper virus for replication. The processing step is RNA-catalysed in all cases, at least in vitro. All plus forms of these RNAs self-cleave via the hammerhead structure whereas only eight of the minus RNAs self-cleave, five via the hammerhead structure and three via the hairpin structure. There are about 20 other viroids where the processing mechanism has yet to be determined but they are likely candidates for a new type of self-cleavage reaction which is predicted to be conserved in all these viroids. Hepatitis delta RNA is the only circular pathogenic RNA known to self-cleave in the animal kingdom. It is feasible that more single-stranded circular pathogenic RNAs are waiting to be discovered and these could be prospective for new types of self-cleavage reactions.
Base Sequence↗
Fluctuations in steady state level of genomic HIV-1 RNA and replication intermediates related to disease progression rate.
Steady state levels of HIV-1 genomic RNA and unintegrated circular DNA fluctuate in the course of HIV-1 infection. Genomic RNA in serum was detected using the NASBA technique and the amount of circular DNA was assessed by PCR. Quantification was done by competitive techniques using co-amplification of internal standards. Within the Amsterdam Cohort Studies it was possible to distinguish rapid progressors, intermediate progressors, slow progressors and non-progressors. Rapid progressors show persistently high viral RNA loads from seroconversion on, while all other HIV-1-infected individuals show a steady decline after seroconversion. Subsequent rises in viral RNA levels herald disease progression in later stages of infection. Unintegrated circular DNA shows similar, but somewhat delayed kinetics. These results indicate that the distribution of AIDS and the average length of the symptom-free period in an HIV-1-infected host population is determined by the steady state levels of genomic RNA and of replication intermediates that are produced by a particular HIV-1 virus population in the average seropositive individual.
Biomarkers↗
Genomic RNAs of influenza viruses are held in a circular conformation in virions and in infected cells by a terminal panhandle.
The viral RNA segments in influenza virions were shown to be circular in conformation by using psoralen crosslinking methods. Electron microscopy of purified RNA following treatment of virus with the psoralen reagent 4'-aminomethyltrioxsalen (AMT) revealed circles with lengths corresponding to the individual segments. RNA blot analysis using polyacrylamide gels demonstrated that RNA from AMT-treated virus had a slowed migration, consistent with it being a single-stranded circle. Furthermore, nuclease S1 protection assays indicated that the termini of the RNA segments form an approximately 15-base-pair-long panhandle. This structure is consistent with the partial sequence complementarity that has been observed for the termini of all influenza virus RNAs. By RNA blot analysis, circular structures of viral sense RNA were also found in influenza virus-infected cells at early and late time points. The circular RNA was the predominant species at the time when the major transcription product is message RNA. This finding and the observation that the termination signal for mRNA synthesis directly abuts the panhandle suggest that a panhandle in the template viral RNA is a cis regulatory signal promoting the synthesis of mRNA instead of plus-sense template. Also, since the panhandle is present in high concentration in virions, we suggest that it is required for packaging and that the input RNA after infection is in the proper conformation for synthesis of primary transcripts.
Cross-Linking Reagents↗
Electron-microscopic mapping of AT-rich regions and of E. coli RNA polymerase-binding sites on the circular kinetoplast DNA of Trypanosoma cruzi.
Partial alkaline denaturation of the circular kinetoplast DNA (kDNA) of Trypanosoma cruzi has shown the existence of 4 small, well-defined AT-rich regions with an average size of about 200 base pairs. They are almost equally distributed, separated by approximately 90 degrees on the circular molecule. All minicircles, whether free or linked in networks, have the same denaturation pattern and, therefore, seem to contain the same information. The long linear molecules present in low amounts in the kDNA samples do not show the same denaturation pattern. Partial denaturation of molecules in larger associations indicates that the circular units may be linked to each other by one strand only. kDNA can be transcribed in vitro by the RNA polymerase of E. coli. RNA polymerase-kDNA complexes have been studied in the electron microscope. By spreading the DNA-protein complexes by adhesion to positively charged carbon films and dark-field observation, it was possible to show the existence of 4 specific binding sites of the E. coli RNA polymerase on the kDNA circles. Comparing the position of the polymerase-binding sites and the AT-rich melted zones, it is suggested that a correlation exists between the two. As had been shown in earlier work, the replication of circular kDNA can be blocked by treating the trypanosomes with the trypanocidal drug Berenil. The comparison of the relative position of the Berenil-blocked replication forks with the position of the 4 denaturation loops shows that the DNA replication is stopped at these AT-rich regions. Since there is evidence that Berenil binds preferentially to AT-rich DNA and seems to be involved in inhibition of DNA replication, the following hypothetical model can be proposed. The replication of the circular kDNA molecules is discontinuous and involves the synthesis of RNA primers; when Berenil is bound to the AT-rich regions, synthesis of new RNA primers is inhibited and replication is blocked at these points, leading to the accumulation of replicating intermediates with defined branch lengths.
Animals↗
RNA-binding strategies common to cold-shock domain- and RNA recognition motif-containing proteins.
Numerous RNA-binding proteins have modular structures, comprising one or several copies of a selective RNA-binding domain generally coupled to an auxiliary domain that binds RNA non-specifically. We have built and compared homology-based models of the cold-shock domain (CSD) of the Xenopus protein, FRGY2, and of the third RNA recognition motif (RRM) of the ubiquitous nucleolar protein, nucleolin. Our model of the CSD(FRG)-RNA complex constitutes the first prediction of the three-dimensional structure of a CSD-RNA complex and is consistent with the hypothesis of a convergent evolution of CSD and RRM towards a related single-stranded RNA-binding surface. Circular dichroism spectroscopy studies have revealed that these RNA-binding domains are capable of orchestrating similar types of RNA conformational change. Our results further show that the respective auxiliary domains, despite their lack of sequence homology, are functionally equivalent and indispensable for modulating the properties of the specific RNA-binding domains. A comparative analysis of FRGY2 and nucleolin C-terminal domains has revealed common structural features representing the signature of a particular type of auxiliary domain, which has co-evolved with the CSD and the RRM.
Amino Acid Motifs↗
Conformation of the RNA polymerase II C-terminal domain: circular dichroism of long and short fragments.
The C-terminal domain (CTD) of the largest subunit of RNA polymerase II consists of tandemly repeated copies of a heptapeptide with the Y(1)S(2)P(3)T(4)S(5)P(6)S(7) consensus sequence. This repeat contains two overlapping SPXX motifs that can adopt a beta-turn conformation. In addition, each CTD repeat contains the PXXP sequence characteristic of the left-handed helix of polyproline II (P(II)) found in SH3 domain ligands and the PXY sequence that is the target for WW domains. We have studied CTD fragments using circular dichroism (CD) to characterize the conformation of the CTD in water and in the hydrogen bond-promoting solvent trifluoroethanol (TFE). In water, an eight-repeat fragment is predominantly unordered, but at 32 degrees C has P(II) and beta-turn contents estimated to be about 15 % and less than 10 %, respectively. In 90 % TFE, the beta-turn fraction is estimated to be about 75 %, the remainder being unordered and P(II) conformations. The Tyr side-chains are ordered to a significant extent in 90 % TFE. Replacement of the fully conserved Pro residues by alpha-aminoisobutyric acid leads to a large increase in beta-turn. Replacement of Ser2 by Ala does not substantially alter the CTD conformation in water or TFE. Ser5 replacement by Ala increases the P(II) content in water and affects the conformation in TFE-rich solutions. Phosphorylation of Ser2 and Ser5 has little effect in water, but Ser2 affects the conformation in TFE-rich solution in much the same way as Ser5-->Ala substitution. The CD of the full-length murine CTD in water is similar to that of the eight-repeat fragment, indicating little difference in conformation with increasing chain length beyond eight repeats. The roles of P(II) and beta-turn in the interaction of CTD with its target proteins (mediator and RNA-processing components) are discussed. The most likely interactions are between P(II) and WW or SH3 domains, or with some unknown P(II)-binding motif.
Amino Acid Motifs↗