A compensatory mutagenesis study of a conserved hairpin in the M gene segment of influenza A virus shows its role in virus replication.

RNA structures are increasingly recognized to be of importance during influenza A virus replication. Here, we investigated a predicted conserved hairpin in the M gene segment (nt 967-994) within the region of the vRNA 5' packaging signal. The existence of this RNA structure and its possible role in virus replication was investigated using a compensatory mutagenesis approach. Mutations were introduced in the hairpin stem, based on natural variation. Virus replication properties were studied for the mutant viruses with disrupted and restored RNA structures. Viruses with structure-disrupting mutations had lower virus titers and a significantly reduced median plaque size when compared with the wild-type (WT) virus, while viruses with structure restoring-mutations replicated comparable to WT. Moreover, virus replication was also reduced when mutations were introduced in the hairpin loop, suggesting its involvement in RNA interactions. Northern blot and FACS experiments were performed to study differences in RNA levels as well as production of M1 and M2 proteins, expressed via alternative splicing. Stem-disruptive mutants caused lower vRNA and M2 mRNA levels and reduced M2 protein production at early time-points. When the RNA structure was restored, vRNA, M2 mRNA and M2 protein levels were increased, demonstrating a compensatory effect. Thus, this study provides evidence for functional importance of the predicted M RNA structure and suggests its role in splicing regulation.

Nemesia ring necrosis virus: a new tymovirus with a genomic RNA having a histidylatable tobamovirus-like 3' end.

The complete nucleotide sequence of the genomic RNA of the new virus Nemesia ring necrosis virus (NeRNV), which is widespread in various ornamental plant species belonging to the Scrophulariaceae and Verbenaceae, has been determined. Based on its gene content, the folding properties of its 5'-untranslated region and in vitro translation experiments, NeRNV RNA is a typical tymovirus RNA. Its 3' end, however, differs greatly from those of the valine-specific tymoviral RNAs that have been analysed previously. It can be folded into an upstream pseudoknot domain and a histidine-specific tRNA-like structure, a combination that, so far, has been found only in tobamoviral RNAs. The identity elements found in NeRNV RNA for recognition by yeast histidyl-tRNA synthetase are more similar to those of yeast tRNAHis than the ones found in tobacco mosaic virus RNA. As a result NeRNV RNA can be charged with histidine even more efficiently than tobacco mosaic virus RNA.

A pH-jump approach for investigating secondary structure refolding kinetics in RNA.

It has been shown that premature translation of the plasmid-mediated toxin in hok/sok of plasmid R1 and pnd/pndB of plasmid R483 is prevented during transcription of the hok and pnd mRNAs by the formation of metastable hairpins at the 5'-end of the mRNA. Here, an experimental approach is presented, which allows the accurate measurement of the refolding kinetics of the 5'-end RNA fragments in vitro without chemically modifying the RNA. The method is based on acid denaturation followed by a pH-jump to neutral pH as a novel way to trap kinetically favoured RNA secondary structures, allowing the measurement of a wide range of biologically relevant refolding rates, with or without the use of standard stopped-flow equipment. The refolding rates from the metastable to the stable conformation in both the hok74 and pnd58 5'-end RNA fragments were determined by using UV absorbance changes corresponding to the structural rearrangements. The measured energy barriers showed that the refolding path does not need complete unfolding of the metastable structures before the formation of the final structures. Two alternative models of such a pathway are discussed.

PseudoBase: structural information on RNA pseudoknots.

PseudoBase is a database containing structural, functional and sequence data related to RNA pseudo-knots. It can be reached at http://wwwbio.LeidenUniv.nl/ approximately Batenburg/PKB.html. For each pseudoknot, thirteen items are stored, for example the relevant sequence, the stem positions of the pseudoknot, the EMBL accession number of the sequence and the support that can be given regarding the reliability of the pseudo-knot. Since the last publication, information on sizes of the stems and the loops in the pseudoknots has been added. Also added are alternative entries that produce surveys of where the pseudoknots are, sorted according to stem size or loop size.

Coupled nucleotide covariations reveal dynamic RNA interaction patterns.

Evolutionarily conserved structures in related RNA molecules contain coordinated variations (covariations) of paired nucleotides. Analysis of covariations is a very powerful approach to deduce phylogenetically conserved (i.e., functional) conformations, including tertiary interactions. Here we discuss conserved RNA folding pathways that are revealed by covariation patterns. In such pathways, structural requirements for alternative pairings cause some nucleotides to covary with two different partners. Such "coupled" covariations between three or more nucleotides were found in various types of RNAs. The analysis of coupled covariations can unravel important features of RNA folding dynamics and improve phylogeny reconstruction in some cases. Importantly, it is necessary to distinguish between multiple covariations determined by mutually exclusive structures and those determined by tertiary contacts.

Genetic manipulation of arterivirus alternative mRNA leader-body junction sites reveals tight regulation of structural protein expression.

To express its structural proteins, the arterivirus Equine arteritis virus (EAV) produces a nested set of six subgenomic (sg) RNA species. These RNA molecules are generated by a mechanism of discontinuous transcription, during which a common leader sequence, representing the 5' end of the genomic RNA, is attached to the bodies of the sg RNAs. The connection between the leader and body parts of an mRNA is formed by a short, conserved sequence element termed the transcription-regulating sequence (TRS), which is present at the 3' end of the leader as well as upstream of each of the structural protein genes. With the exception of RNA3, only one body TRS was previously assumed to be used to join the leader and body of each EAV sg RNA. Here we show that for the synthesis of two other sg RNAs, RNA4 and RNA5, alternative leader-body junction sites that differ substantially in transcriptional activity are used. By site-directed mutagenesis of an EAV infectious cDNA clone, the alternative TRSs used to generate RNA3, -4, and -5 were inactivated, which strongly influenced the corresponding RNA levels and the production of infectious progeny virus. The relative amounts of RNA produced from alternative TRSs differed significantly and corresponded to the relative infectivities of the virus mutants. This strongly suggested that the structural proteins that are expressed from these RNAs are limiting factors during the viral life cycle and that the discontinuous step in sg RNA synthesis is crucial for the regulation of their expression. On the basis of a theoretical analysis of the predicted RNA structure of the 3' end of the EAV genome, we propose that the local secondary RNA structure of the body TRS regions is an important factor in the regulation of the discontinuous step in EAV sg mRNA synthesis.

PseudoBase: a database with RNA pseudoknots.

PseudoBase is a database containing structural, functional and sequence data related to RNA pseudo-knots. It can be reached at http://wwwbio. Leiden Univ.nl/ approximately Batenburg/PKB.html. This page will direct the user to a retrieval page from where a particular pseudoknot can be chosen, or to a submission page which enables the user to add pseudoknot information to the database or to an informative page that elaborates on the various aspects of the database. For each pseudoknot, 12 items are stored, e.g. the nucleotides of the region that contains the pseudoknot, the stem positions of the pseudoknot, the EMBL accession number of the sequence that contains this pseudoknot and the support that can be given regarding the reliability of the pseudoknot. Access is via a small number of steps, using 16 different categories. The development process was done by applying the evolutionary methodology for software development rather than by applying the methodology of the classical waterfall model or the more modern spiral model.

Metastable structures and refolding kinetics in hok mRNA of plasmid R1.

Programmed cell death by hok/sok of plasmid R1 and pnd/pndB of R483 mediates plasmid maintenance by killing of plasmid-free cells. It has been previously suggested that premature translation of the plasmid-mediated toxin is prevented during transcription of the hok and pnd mRNAs by the formation of metastable hairpins in the mRNA at the 5' end. Here, experimental evidence is presented for the existence of metastable structures in the 5' leader of the hok and pnd mRNAs in vitro. The kinetics of refolding from the metastable to the stable structure in the isolated fragments of the 5' ends of both the hok and pnd mRNAs could be estimated, in agreement with the structural rearrangement in this region, as predicted to occur during transcription and mRNA activation. The refolding rates of hok and pnd structures are slow enough to allow for the formation of downstream hairpin structures during elongation of the mRNAs, which thereby helps to stabilize the metastable structures. Thus, the kinetic refolding parameters of the hok and pnd mRNAs are consistent with the proposal that the metastable structures prevent premature translation and/or antisense RNA binding during transcription.

Arterivirus discontinuous mRNA transcription is guided by base pairing between sense and antisense transcription-regulating sequences.

To generate an extensive set of subgenomic (sg) mRNAs, nidoviruses (arteriviruses and coronaviruses) use a mechanism of discontinuous transcription. During this process, mRNAs are generated that represent the genomic 5' sequence, the so-called leader RNA, fused at specific positions to different 3' regions of the genome. The fusion of the leader to the mRNA bodies occurs at a short, conserved sequence element, the transcription-regulating sequence (TRS), which precedes every transcription unit in the genome and is also present at the 3' end of the leader sequence. Here, we have used site-directed mutagenesis of the infectious cDNA clone of the arterivirus equine arteritis virus to show that sg mRNA synthesis requires a base-pairing interaction between the leader TRS and the complement of a body TRS in the viral negative strand. Mutagenesis of the body TRS of equine arteritis virus RNA7 reduced sg RNA7 transcription severely or abolished it completely. Mutations in the leader TRS dramatically influenced the synthesis of all sg mRNAs. The construction of double mutants in which a mutant leader TRS was combined with the corresponding mutant RNA7 body TRS resulted in the specific restoration of mRNA7 synthesis. The analysis of the mRNA leader-body junctions of a number of mutants with partial transcriptional activity provided support for a mechanism of discontinuous minus-strand transcription that resembles similarity-assisted, copy-choice RNA recombination.

An approximation of loop free energy values of RNA H-pseudoknots.

A set of free energy values is suggested for RNA H-pseudoknot loops. The parameters are adjusted to be consistent with the theory of polymer thermodynamics and known data on pseudoknots. The values can be used for estimates of pseudoknot stabilities and computer predictions of RNA structures.

The mechanism of actinomycin D-mediated inhibition of HIV-1 reverse transcription.

The mechanism of reverse transcription was analyzed in vitro with RNA templates and the reverse transcriptase (RT) enzyme of human immunodeficiency virus type 1 (HIV-1). In particular, we analyzed the mechanism of actinomycin D (ActD) mediated inhibition of the strand transfer step, in which the newly synthesized cDNA, termed the (-) strand strong stop or (-)ssDNA, is transferred from the donor RNA onto the acceptor RNA. This strand transfer reaction is a rather inefficient process in vitro. We found that this is in part due to the presence of an excess donor RNA, and highly efficient strand transfer was achieved by reducing the amount of donor RNA. We suggest that annealing of the (-)ssDNA to the excess donor RNA is preferred over productive binding to the acceptor RNA because of a higher basepair complementarity. ActD remains a potent inhibitor of strand transfer in this optimized assay system. We measured no effect of ActD on the elongation of reverse transcription or the RNase H action of the RT enzyme. Instead, we provide evidence that ActD acts through direct interaction with the (-)ssDNA, thereby blocking the basepairing capacity of this molecule. The possible use of single-stranded DNA binding molecules as antiretroviral agents is discussed.

The nucleotide sequence of satellite tobacco necrosis virus strain C and helper-assisted replication of wild-type and mutant clones of the virus.

The complete nucleotide sequence of satellite tobacco necrosis virus strain C (STNV-C) was determined. The genome has a similar overall organization to two STNV isolates studied previously but differs significantly from them in the secondary structure of the translated and untranslated regions (UTRs). STNV-C RNA is naturally uncapped and contains 1221 nt: 101 nt in the 5' UTR, 606 nt in the capsid protein (CP) coding region and 514 nt in the 3' UTR. Using the known sequences of STNV-C and tobacco necrosis virus strain D (TNV-D) RNAs, full-length cDNA clones of both RNAs were constructed. Synthetic transcripts derived from STNV-C cDNA clones only replicated in plants and protoplasts when co-inoculated with TNV-D transcripts. A number of mutant clones in both the 3' and the 5' STNV-C RNA UTRs were constructed which disrupted putative cis-acting elements recognized by helper virus polymerase. Deletion analysis revealed an essential requirement of all 3' and 5' proximal sequences in the STNV-C UTRs for replication. However, an internal region in the 3' UTR could be deleted without loss of infectivity. Likewise, the entire STNV-C CP-encoding region could be deleted and replaced with a marker gene of a similar size without loss of transcript accumulation in plants.

Dynamic competition between alternative structures in viroid RNAs simulated by an RNA folding algorithm.

The folding pathways of viroid RNAs were studied using computer simulations by the genetic algorithm for RNA folding. The folding simulations were performed for PSTVd RNAs of both polarities, using the wild-type sequence and some previously known mutants with suggested changes in the stable or metastable structures. It is shown that metastable multihairpin foldings in the minus strand replicative intermediates are established due to the specific folding pathway that ensures the absence of the most stable rod-like structure. Simulations of the PSTVd minus strand folding during transcription reveal a metastable hairpin, formed in the left terminal domain region of the PSTVd. Despite high sequence variability, this hairpin is conserved in all known large viroids of both subgroups of PSTVd type, and is presumably necessary to guide the folding of the HPII hairpin which is functional in the minus strand. The folding simulations are able to demonstrate the changes in the balance between metastable and stable structures in mutant PSTVd RNAs. The stable rod-like structure of the circular viroid (+) RNA is also folded via a dynamic folding pathway. Furthermore, the simulations show that intermediate steps in the forced evolution of a shortened PSTVd replicon may be reconstructed by a mechanistic model of different folding pathway requirements in plus- and minus-strand RNAs. Thus the formation of viroid RNA structure strongly depends on dynamics of competition between alternative RNA structures. This also suggests that the replication efficiency of viroid sequences may be estimated by a simulation of the folding process.

Programmed cell death by hok/sok of plasmid R1: coupled nucleotide covariations reveal a phylogenetically conserved folding pathway in the hok family of mRNAs.

The hok/sok system of plasmid R1 mediates plasmid maintenance by killing of plasmid-free cells. Translation of the stable toxin-encoding hok mRNA is repressed by the unstable Sok antisense RNA. Using genetic algorithm simulations and phylogenetic comparisons, we analyse five plasmid-encoded and two chromosome-encoded hok-homologous mRNAs. A similar folding pathway was found for all mRNAs. Metastable hairpins at the very 5'-ends of the mRNAs were predicted to prevent the formation of structures required for translation and antisense RNA binding. Thus the folding of the mRNA 5'-ends appears to explain the apparent inactivity of the nascent transcripts. In the full-length mRNAs, long-range 5' to 3' interactions were predicted in all cases. The 5' to 3' interactions lock the mRNAs in inactive configurations. Translation of the mRNAs is activated by 3' exonucleolytic processing. Simulation of the 3' processing predicted that it triggers rearrangements of the mRNA 5'-ends with the formation of translational activator and antisense RNA target hairpins. Alignment of the mRNA sequences revealed a large number of nucleotide covariations that support the existence of the proposed secondary structures. Furthermore, coupled covariations support the folding pathway and provide evidence that the mRNA 5'-ends pair with three different partners during the proposed series of dynamic RNA rearrangements.

Programmed cell death by hok/sok of plasmid R1: processing at the hok mRNA 3'-end triggers structural rearrangements that allow translation and antisense RNA binding.

The hok/sok locus of plasmid R1 mediates plasmid stabilization by killing of plasmid-free cells. The locus specifies two RNAs, hok mRNA and Sok antisense RNA. The post-segregational killing mediated by hok/sok is governed by a complicated control mechanism that involves both post-transcriptional inhibition of translation by Sok-RNA and activation of hok translation by mRNA 3' processing. Sok-RNA inhibits translation of a reading frame (mok) that overlaps with hok, and translation of hok is coupled to translation of mok. In the inactive full-length hok mRNA, the translational activator element at the mRNA 5'-end (tac) is sequestered by the fold-back-inhibitory element located at the mRNA 3'-end (fbi). The 5' to 3' pairing locks the RNA in an inert configuration in which the SDmok and Sok-RNA target regions are sequestered. Here we show that the 3' processing leads to major structural rearrangements in the mRNA 5'-end. The structure of the refolded RNA explains activation of translation and antisense RNA binding. The refolded RNA contains an antisense RNA target stem-loop that presents the target nucleotides in a single-stranded conformation. The stem of the target hairpin contains SDmok and AUGmok in a paired configuration. Using toeprinting analysis, we show that this pairing keeps SDmok in an accessible configuration. Furthermore, a mutational analysis shows that an internal loop in the target stem is prerequisite for efficient translation and antisense RNA binding.

Antisense RNA-regulated programmed cell death.

Eubacterial plasmids and chromosomes encode multiple killer genes belonging to the hok gene family. The plasmid-encoded killer genes mediate plasmid stabilization by killing plasmid-free cells. This review describes the genetics, molecular biology, and evolution of the hok gene family. The complicated antisense RNA-regulated control-loop that regulates posttranscriptional and postsegregational activation of killer mRNA translation in plasmid-free cells is described in detail. Nucleotide covariations in the mRNAs reveal metastable stem-loop structures that are formed at the mRNA 5' ends in the nascent transcripts. The metastable structures prevent translation and antisense RNA binding during transcription. Coupled nucleotide covariations provide evidence for a phylogenetically conserved mRNA folding pathway that involves sequential dynamic RNA rearrangements. Our analyses have elucidated an intricate mechanism by which translation of an antisense RNA-regulated mRNA can be conditionally activated. The complex phylogenetic relationships of the plasmid- and chromosome-encoded systems are also presented and discussed.

Secondary structure model of the coat protein gene of turnip yellow mosaic virus RNA: long, C-rich, single-stranded regions.

The RNA of all tymoviruses, a group of ssRNA plant viruses, has a base composition that is different from that of most other viruses. The excess of cytosines (35-42%) and the low number of guanosines (15-17%) must impel an RNA structure with a relatively low amount of base pairing and a high incidence of unpaired cytosines. These unpaired cytosines probably function in RNA-protein interactions. To gain Insight into the way the RNA is positioned inside the virion, the secondary structure has been determined of a part of TYMV RNA, including the so-called tymobox, the coat protein gene, and the 3' untranslated region, by structure probing, sequence comparison, and computer predictions. Conservation of secondary structure elements in tymoviruses is not high and does not parallel the conservation of the primary structure. A combination of structure prediction and probing experiments, however, results in a model consisting of structured domains of 100-200 nucleotides interspersed by long unpaired cytosine-rich regions. The latter may interact with the coat protein inside the virion. The structure of some functionally interesting regions of the 3' part of TYMV RNA is also discussed.

Phylogenetic relationships inferred from the sequence and secondary structure of ITS1 rRNA in Albinaria and putative Isabellaria species (Gastropoda, Pulmonata, Clausiliidae).

An analysis of the ITS1 sequence variation among five species of terrestrial pulmonate snails was performed to decide between two conflicting hypotheses concerning the phylogeny of these anatomically similar gastropods. It turned out that the so-called genus Isabellaria is a polyphyletic entity; the diagnostic, apomorphic structure of its clausilial apparatus, enabling a nearly complete obstruction of the shell aperture with the animal at rest, apparently evolved more than once from ancestors currently classified with the speciose genus Albinaria. The classification based on general shell shape and sculpture, and distributional patterns, turns out to be the natural one. This study also provides the first data on ITS1 sequences in gastropods. The recently published ITS1 sequence of another molluscan species, a bivalve, is quite different but similar in length to that of the snails.

The influence of a metastable structure in plasmid primer RNA on antisense RNA binding kinetics.

Replication of the ColE1 group plasmids is kinetically regulated by the interaction between plasmid-encoded primer RNA II and antisense RNA I. The binding is dependent on alternative RNA II conformations, formed during the transcription, and effectively inhibits the primer function within some time interval. In this paper, the folding pathways for the wild type and copy number mutants of ColE1 RNA II are studied using simulations by a genetic algorithm. The simulated pathways reveal a transient formation of a metastable structure, which is stabilized by copy number mutations. The folding kinetics of the proposed conformational transitions is calculated using a model of a multistep refolding process with elementary steps of double-helical stem formation or disruption. The approximation shows that the lifetime of the metastable structure is relatively long and is considerably increased in the mutants, resulting in a delay of the formation of the stable RNA II structure, which is the most sensitive to the inhibition by the antisense RNA I. Thus the effect of copy number mutations can be interpreted as a compression of the time window of effective inhibition due to an increased time spent by the RNA II in the metastable state. The implications of metastable foldings in RNA functioning are discussed.

The computer simulation of RNA folding pathways using a genetic algorithm.

A procedure for simulating the RNA folding process using the principles of genetic algorithm is proposed. The method allows one to simulate a folding pathway of RNA, including such processes as disruption of temporarily formed structures, the folding of a molecule during its synthesis and pseudoknot formation. The simulations are able to predict functional metastable foldings and kinetically driven transitions to more stable structures. The analysis of free energies for intermediate foldings allows estimation of the ranges of kinetic refolding barriers and suggests that in some RNAs the selective evolutionary pressure suppresses the possibilities for alternative structures that could form in the course of transcription. It is shown that the folding pathway simulation can result in structure predictions that are more consistent with phylogenetically proven structures than minimum energy solutions. This suggest that RNA folding kinetics is very important for the formation of functional RNA structures. Therefore, apart form its value for predictions of RNA structures, the proposed computer simulations can be a powerful tool in the studies of RNA folding features.