Application of circular dichroism to study RNA folding transitions.
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Circular Dichroism↗
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Two reactions of Haloferax volcanii RNA splicing enzymes: joining of exons and circularization of introns.
Archaeal RNA splicing involves at least two protein enzymes, a specific endonuclease and a specific ligase. The endonuclease recognizes and cleaves within a characteristic bulge-helix-bulge (BHB) structure formed by pairing of the regions near the two exon-intron junctions, producing 2',3'-cyclic phosphate and 5'-hydroxyl termini. The ligase joins the exons and converts the cyclic phosphate into junction phosphate. The ligated product contains a seven-base hairpin loop, in which the splice junction is in between the two 3' terminal residues of the loop. Archaeal splicing endonucleases are also involved in rRNA processing, cutting within the BHB structures formed by pairing of the 5' and 3' flanking regions of the rRNAs. Large free introns derived from pre-rRNAs have been observed as stable and abundant circular RNAs in certain Crenarchaeota, a kingdom in the domain Archaea. In the present study, we show that the cells of Haloferax volcanii, a Euryarchaeote, contain circular RNAs formed by 3',5'-phosphodiester linkage between the two termini of the introns derived from their pre-tRNAs. H. volcanii ligase, in vitro, can also circularize both endonuclease-cleaved introns, and non-endonuclease-produced substrates. Exon joining and intron circularization are mechanistically similar ligation reactions that can occur independently. The size of the ligated hairpin loop and position of the splice junction within this loop can be changed in in vitro ligation reactions. Overall, archaeal RNA splicing seems to involve two sets of two symmetric transesterification reactions each.
Exons↗
Splicing of a circular yeast pre-mRNA in vitro.
We have tested the fate of a circularized synthetic pre-mRNA transcript in a whole cell splicing extract of Saccharomyces cerevisiae. Our results demonstrate that this circular precursor RNA is able to induce spliceosome formation in vitro and that the products of the following splicing reaction are the lariat-shaped intron, and a mature circular mRNA. Thus, it would appear that free 5' and/or 3' ends are not obligatory for a splicing reaction to occur, although we find its efficiency to be strongly influenced by the presence or lack of free ends. To our knowledge, this is the first demonstration that a circular pre-mRNA molecule is recognized as a suitable substrate by an eukaryotic mRNA splicing apparatus.
Base Sequence↗
Characterization of multiple circular RNAs derived from a plant viroid-like RNA by sequence deletions and duplications.
Northern blot hybridizations with a probe complementary to a 275-nt circular RNA isolated from carnation plants revealed that this RNA co-exists in vivo with minor amounts of other small circular RNAs. Sequencing of cDNA clones of nine RNA species demonstrated deletions and duplications of the predominant 275-nt RNA. Minor sequence heterogeneities were observed at the crossover sites. Deletions mapped to three arms of the cruciform structure of lowest free energy obtained previously for the parental 275-nt RNA, whereas repeats encompassed mostly regions of the arm where deletions were not found. Some of the deleted and duplicated regions corresponded to sequences forming part of the two hammerhead structures involved in the in vitro self-cleavage of the plus and minus strands of the 275-nt RNA. A copy choice model is proposed for the emergence of deletions and duplications, where the RNA polymerase with the nascent strand dissociates from the template at regions rich in secondary and possibly tertiary structures, and reinitiates synthesis at different upstream and downstream positions.
Base Sequence↗
Internal ribosome entry sites tests with circular mRNAs.
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Humans↗
Yeast RNA polymerase II transcription of circular DNA at different degrees of supercoiling.
Purified yeast RNA polymerase II was tested for transcriptional activity as a function of the degree of circular DNA supercoiling. Chimaeric plasmids P30 and P31 both containing inserts from the yeast transposable element TY1 cloned in pBR322 and the vector pBR322 were used as templates. For pBR322 the transcriptional activity increases about 4 fold from the fully relaxed covalently closed circles to the native supercoiled forms, further supercoiling having no effect on transcription. P30 shows a 5 fold increase of transcriptional activity reaching a plateau at the native supercoiled conformation. However, at an intermediate degree of supercoiling (sigma = 0.024), transcription decreases to a value close to zero. P31 too exhibits a conformation (sigma = 0.014) in which there is a drop of transcriptional activity. Furthermore, a 10 fold increase of transcription is obtained at the higher values of superhelix density. Both kinetic and autoradiographic experiments confirm the existence of DNA conformations that can inhibit "in vitro" transcription.
DNA Transposable Elements↗
In vitro synthesis of an infectious viroid: analysis of the infectivity of monomeric linear CEV.
Infectious monomers of citrus exocortis viroid (CEV) were synthesized in vitro precisely to predetermined sequences in microgram quantities without resorting to cloning procedures. Amplification of CEV double-stranded cDNAs fused with a T7 RNA polymerase promoter was followed by transcription of the DNA resulting in the production of an infectious linear CEV monomer. This is the first demonstration of an infectious unit length viroid synthesized in vitro. Transcripts containing 3'-OH terminal groups were infectious, demonstrating that a 2',3'-cyclic phosphate terminus is not a prerequisite for viroid infectivity as previously suggested. Conversion of the 5'-triphosphate terminus to either 5'-monophosphate or 5'-OH had little effect on infectivity. The linear RNA could be circularized using T4 RNA ligase to produce an authentic CEV molecule. This procedure, which results in the production of biologically active RNA, would allow routine application of oligonucleotide-directed mutagenesis to the study of viroids and other circular RNAs. It would also enable the in vitro synthesis and mutagenesis of infectious viral RNAs containing a 5'-G residue.
Base Sequence↗
Synthesis and hybridization properties of RNA containing 8-chloroadenosine.
8-Chloroadenosine (8-Cl-Ado) has shown potential as a chemotherapeutic agent for the treatment of multiple myeloma and certain leukemias. 8-Cl-Ado treatment leads to a decrease in global RNA levels and incorporation of the analog into cellular RNA in malignant cells. To investigate the effects of 8-Cl-Ado modifications on RNA structure and function, an 8-Cl-Ado phosphoramidite and controlled-pore glass support were synthesized and used to introduce 8-Cl-Ado at internal and 3'- terminal positions, respectively. RNA oligonucleotides containing 8-chloroadenine (8-Cl-A) residues were synthesized and hybridized with complementary RNA strands. Circular dichroism spectroscopy of the resulting RNA duplexes revealed that the modified nucleobase does not perturb the overall A-form helix geometry. The thermal stabilities of 8-Cl-Ado modified duplexes were determined by UV thermal denaturation analysis and were compared with analogous natural duplexes containing standard and mismatched base pairs. The 8-Cl-Ado modification destabilizes RNA duplexes by approximately 5 kcal/mole, approximately as much as a U:U mismatched base pair. The duplex destabilization of 8-Cl-A may result from perturbation of Watson-Crick base pairing induced by conformational preferences of 8-halogenated nucleosides.
2-Chloroadenosine↗
Intracellular cleavage and ligation of hepatitis delta virus genomic RNA: regulation of ribozyme activity by cis-acting sequences and host factors.
During replication, a ribozyme within the genomic RNA of hepatitis delta virus cleaves multimeric precursors to release a unit-length linear intermediate. Intramolecular ligation of this intermediate produces the circular genomic RNA. Although one copy of the ribozyme is reconstituted by such ligation, it does not subsequently cleave and destroy the circular conformation. We have identified cis-acting attenuator sequences that prevent self-cleavage of the circular product by base pairing with and inactivating the ribozyme. Furthermore, we have shown that during the initial processing of the multimeric precursor RNA, host-specific factors activate the ribozyme by preventing its association with the attenuator sequences. Thus, we demonstrate a novel switching mechanism that regulates ribozyme activity inside the cell.
Base Sequence↗
Ribonuclease E is a 5'-end-dependent endonuclease.
The selective degradation of messenger RNAs enables cells to regulate the levels of particular mRNAs in response to changes in the environment. Ribonuclease (RNase) E, a single-strand-specific endonuclease that is found in a multi-enzyme complex known as the 'degradosome', initiates the degradation of many mRNAs in Escherichia coli. Its relative lack of sequence specificity and the presence of many potential cleavage sites in mRNA substrates cannot explain why mRNA decay frequently proceeds in a net 5'-to-3' direction. I have prepared covalently closed circular derivatives of natural substrates, the rpsT mRNA encoding ribosomal protein S20 and the 9S precursor to 5S ribosomal RNA, and find that these derivatives are considerably more resistant to cleavage in vitro by RNase E than are linear molecules. Moreover, antisense oligo-deoxynucleotides complementary to the 5' end of linear substrates significantly reduce the latter's susceptibility to attack by RNase E. Finally, natural substrates with terminal 5'-triphosphate groups are poorly cleaved by RNase E in vitro, whereas 5' monophosphorylated substrates are strongly preferred. These results show that RNase E has inherent vectorial properties, with its activity depending on the 5' end of its substrates; this can account for the direction of mRNA decay in E. coli, the phenomenon of 'all or none' mRNA decay, and the stabilization provided by 5' stem-loop structures.
Binding Sites↗
Folding of circularly permuted transfer RNAs.
All of the ribose-phosphate linkages in yeast tRNA(Phe) that could be cleaved without affecting the folding of the molecule have been determined in a single experiment. Circular permutation analysis subjects circular tRNA molecules to limited alkaline hydrolysis in order to generate one random break per molecule. Correctly folded tRNAs were identified by lead cleavage at neutral pH, a well-characterized reaction that requires proper folding of tRNA(Phe). Surprisingly, most of the circularly permuted tRNA molecules folded correctly. This result suggests that the tRNA folding motif could occur internally within other RNA sequences, and a computer search of Genbank entries has identified many examples of such motifs.
Base Sequence↗
Evidence for Z-form RNA by vacuum UV circular dichroism.
Circular dichroism (CD) spectra in the vacuum UV region for different conformations of poly d(G-C) X poly d(G-C) and poly r(G-C) X poly r(G-C) are very characteristic. The CD of the RNA in the A-form (6 M NaClO4 and 22 degrees C) is very similar to that of the DNA in 80% alcohol where it is believed to be in the A-form. With the exception of the longest wavelength transition, the CD of the RNA in 6 M NaClO4 at 46 degrees C is similar to the CD of the DNA under conditions where it is believed to be in the Z-form (2 M NaClO4). This substantiates that poly r(G-C) X poly r(G-C) assumes a left-handed Z-conformation in 6 M NaClO4 above 35 degrees C. CD spectra for the left-handed Z-forms of both the RNA and DNA are characterized by an intense negative peak at 190-195 nm, a crossover at about 184 nm, and an intense positive peak below 180 nm. The right-handed A- and B-forms of RNA and DNA all have an intense positive peak in their CD spectra near 186 nm. The large difference in CD in the range 185-195 nm for right- and left-handed conformations of nucleic acids can be used to identify the sense of helix winding.
Circular Dichroism↗
Circularization of linear viroid RNA via 2'-phosphomonoester, 3', 5'-phosphodiester bonds by a novel type of RNA ligase from wheat germ and Chlamydomonas.
A novel type of RNA ligase activity in extracts of wheat germ or Chlamydomonas requires 2', 3'-cyclic phosphate and 5'-phosphate ends for ligation to form a 2'-phosphomonoester, 3',5'-phosphodiester bond. Using 5'-3 2P-labeled linear PSTV, we demonstrate that RNase T1-nicked viroid predominantly forms (formula; see text) U-bonds. Natural linear PSTV, however, forms mainly (formula; see text) A-bonds upon enzymatic circularization. We show that natural linear PSTV RNA has nicks between C181 and A182, or between C348 and A349, and that consequently C181 and C348 carry 2',3'-cyclophosphate termini.
Chemical Phenomena↗
A general strategy for cloning viroids and other small circular RNAs that uses minimal amounts of template and does not require prior knowledge of its sequence.
Two PCR-based methods are described for obtaining clones of small circular RNAs of unknown sequence and for which only minute amounts are available. To avoid introducing any assumption about the RNA sequence, synthesis of the cDNAs is initiated with random primers. The cDNA population is then PCR-amplified using a primer whose sequence is present at both sides of the cDNAs, since they have been obtained with random hexamers and then a linker with the sequence of the PCR primer has been ligated to their termini, or because the cDNAs have been synthesized with an oligonucleotide that contains the sequence of the PCR primer at its 5' end and six randomized positions at its 3' end. The procedures need only approximately 50 ng of purified RNA template. The reasons for the emergence of cloning artifacts and precautions to avoid them are discussed.
Base Sequence↗
Inability of circular mRNA to attach to eukaryotic ribosomes.
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Eukaryotic Cells↗
Initiation of protein synthesis by the eukaryotic translational apparatus on circular RNAs.
The ribosome scanning model predicts that eukaryotic ribosomal 40S subunits enter all messenger RNAs at their 5' ends. Here, it is reported that eukaryotic ribosomes can initiate translation on circular RNAs, but only if the RNAs contain internal ribosome entry site elements. Long-repeating polypeptide chains were synthesized from RNA circles with continuous open reading frames. These results indicate that ribosomes can translate such RNA circles for multiple consecutive rounds and that the free 5' end of a messenger RNA is not necessarily the entry point for 40S subunits.
Codon↗
Selection of circularization sites in a group I IVS RNA requires multiple alignments of an internal template-like sequence.
Circularization and reverse circularization of the Tetrahymena thermophila rRNA intervening sequence resemble the first and second steps in splicing, respectively. However, site-specific base substitutions show that different nucleotides are involved in selection of the 5' splice site and the circularization sites. Furthermore, a substitution at the major circularization site that prevents circularization can be suppressed by second substitutions at two different nucleotide positions. A model is proposed in which adjacent and overlapping sequences can function as a binding site, forming a short duplex with the sequence at the circularization site and thus directing circularization and reverse circularization. Because the 5' exon-binding site and three potential circularization binding sites fall within a contiguous eight nucleotide region, this sequence may translocate relative to the catalytic core of the ribozyme in a template-like manner.
Animals↗
In vitro transcription by wheat germ RNA polymerase II. Initiation of RNA synthesis on relaxed, closed circular template.
We have developed a novel method for studying the ability to wheat germ RNA polymerase II to initiate transcription in vitro on unnicked duplex DNA template. Our template is a plasmid which has been relaxed in vitro with wheat germ DNA topoisomerase I. The plasmid consists of SV40 cloned in pBR322. We transcribe briefly to synthesize highly labeled nascent RNA, then fractionate the resulting ternary complexes on an agarose gel to separate relaxed, closed circular DNA from other DNA species contaminating the template preparation. Under optimal conditions, we recover about half of the total RNA synthesized in complex with relaxed, closed circular DNA. The remaining RNA is in association with other DNA species or is free in solution. The principal conclusions we draw from these studies are: 1) relaxed, closed circular DNA can serve as a template for in vitro transcription, although the relative efficiencies of transcription are such that even a few per cent contamination with nicked or supercoiled template can cause transcription from these forms to dominate the reaction. 2) Initiation occurs on approximately 1 in 25 cloned circular molecules in the course of a 5-min reaction, under optimal conditions. This transcription is not predominantly from SV40 in vivo promoters and is either random or from a sufficient number of sites as to appear random. 3) There is a striking dependence of initiation frequency on purine nucleoside triphosphate concentration, especially with GTP.
DNA Topoisomerases, Type I↗