Viral RNA pseudoknots: versatile motifs in gene expression and replication.
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ABSTRACT:
SUBMITTER: Brierley I
PROVIDER: S-EPMC7096944 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:MotivationThe prediction of RNA structure canonical base pairs from a single sequence, especially pseudoknotted ones, remains challenging in a thermodynamic models that approximates the energy of the local 3D motifs joining canonical stems. It has become more and more apparent in recent years that the structural motifs in the loops, composed of noncanonical interactions, are essential for the final shape of the molecule enabling its multiple functions. Our capacity to predict accurate 3D structures is also limited when it comes to the organization of the large intricate network of interactions that form inside those loops.ResultsWe previously developed the integer programming framework RNA Motifs over Integer Programming (RNAMoIP) to reconcile RNA secondary structure and local 3D motif information available in databases. We further develop our model to now simultaneously predict the canonical base pairs (with pseudoknots) from base pair probability matrices with or without alignment. We benchmarked our new method over the all nonredundant RNAs below 150 nucleotides. We show that the joined prediction of canonical base pairs structure and local conserved motifs (i) improves the ratio of well-predicted interactions in the secondary structure, (ii) predicts well canonical and Wobble pairs at the location where motifs are inserted, (iii) is greatly improved with evolutionary information, and (iv) noncanonical motifs at kink-turn locations.Availability and implementationThe source code of the framework is available at https://gitlab.info.uqam.ca/cbe/RNAMoIP and an interactive web server at https://rnamoip.cbe.uqam.ca/.
| S-EPMC10868335 | biostudies-literature
Project description:The RNA genomes from the cardioviruses, hepatoviruses, and aphthoviruses encode two to five tandem pseudoknots within their 5' untranslated regions. These pseudoknots lie adjacent to a pyrimidine-rich sequence, which in cardio- and aphthoviruses takes the form of a homopolymeric poly(C) tract. Seven deletion mutations within mengovirus pseudoknots PK(B) and PK(C) were created and characterized. tested in tissue culture, mengovirus genomes with alterations in PK(C) were viable but had small plaque phenotypes. Larger plaque revertants were isolated and partially characterized, and each proved to be a second-site pseudorevertant with (unmapped) changes elsewhere in the genome. The infectious PK(C) mutant viruses were highly lethal to mice, and deletions in this motif did not affect mengovirus virulence in the same manner as deletions in the adjacent poly(C) tract. In contrast, deletions in PK(B), or deletions which spanned PK(B) + PK(C), produced nonviable genomes. Cell-free translations directed by any of the altered PK sequences gave normal polyprotein amounts relative to wild-type mengovirus. But viral RNA accumulation during HeLa cell infection was dramatically impaired, even with the least disruptive of the PK(C) changes, suggesting the pseudoknots play an essential though undefined role in RNA synthesis and moreover that an intact PK(B) structure is critical to this function.
| S-EPMC190899 | biostudies-other
Project description:RNA pseudoknots are important for function. Three-dimensional structural information is available, insights into factors affecting pseudoknot stability are being reported, and computer programs are available for predicting pseudoknots.
| S-EPMC2873773 | biostudies-literature
Project description:MotivationThe function of an RNA molecule is not only linked to its native structure, which is usually taken to be the ground state of its folding landscape, but also in many cases crucially depends on the details of the folding pathways such as stable folding intermediates or the timing of the folding process itself. To model and understand these processes, it is necessary to go beyond ground state structures. The study of rugged RNA folding landscapes holds the key to answer these questions. Efficient coarse-graining methods are required to reduce the intractably vast energy landscapes into condensed representations such as barrier trees or basin hopping graphs : BHG) that convey an approximate but comprehensive picture of the folding kinetics. So far, exact and heuristic coarse-graining methods have been mostly restricted to the pseudoknot-free secondary structures. Pseudoknots, which are common motifs and have been repeatedly hypothesized to play an important role in guiding folding trajectories, were usually excluded.ResultsWe generalize the BHG framework to include pseudoknotted RNA structures and systematically study the differences in predicted folding behavior depending on whether pseudoknotted structures are allowed to occur as folding intermediates or not. We observe that RNAs with pseudoknotted ground state structures tend to have more pseudoknotted folding intermediates than RNAs with pseudoknot-free ground state structures. The occurrence and influence of pseudoknotted intermediates on the folding pathway, however, appear to depend very strongly on the individual RNAs so that no general rule can be inferred.Availability and implementationThe algorithms described here are implemented in C++ as standalone programs. Its source code and Supplemental material can be freely downloaded from http://www.tbi.univie.ac.at/bhg.html.Contactqin@bioinf.uni-leipzig.deSupplementary informationSupplementary data are available at Bioinformatics online.
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Project description:Natural RNA molecules can have a high degree of structural complexity but even the most complexly folded RNAs are assembled from simple structural building blocks. Among the simplest RNA elements are double-stranded helices that participate in the formation of different folding topologies and constitute the major fraction of RNA structures. One common folding motif of RNA is a pseudoknot, defined as a bipartite helical structure formed by base-pairing of the apical loop in the stem-loop structure with an outside sequence. Pseudoknots constitute integral parts of the RNA structures essential for various cellular activities. Among many functions of pseudoknotted RNAs is feedback regulation of gene expression, carried out through specific recognition of various molecules. Pseudoknotted RNAs autoregulate ribosomal and phage protein genes in response to downstream encoded proteins, while many metabolic and transport genes are controlled by cellular metabolites interacting with pseudoknotted RNA elements from the riboswitch family. Modulation of some genes also depends on metabolite-induced messenger RNA (mRNA) cleavage performed by pseudoknotted ribozymes. Several regulatory pseudoknots have been characterized biochemically and structurally in great detail. These studies have demonstrated a plethora of pseudoknot-based folds and have begun uncovering diverse molecular principles of the ligand-dependent gene expression control. The pseudoknot-mediated mechanisms of gene control and many unexpected and interesting features of the regulatory pseudoknots have significantly advanced our understanding of the genetic circuits and laid the foundation for modulation of their outcomes.
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Project description:Programmed ribosomal frameshifting (PRF) is one of the multiple translational recoding processes that fundamentally alters triplet decoding of the messenger RNA by the elongating ribosome. The ability of the ribosome to change translational reading frames in the -1 direction (-1 PRF) is employed by many positive strand RNA viruses, including economically important plant viruses and many human pathogens, such as retroviruses, e.g., HIV-1, and coronaviruses, e.g., the causative agent of severe acute respiratory syndrome (SARS), in order to properly express their genomes. -1 PRF is programmed by a bipartite signal embedded in the mRNA and includes a heptanucleotide "slip site" over which the paused ribosome "backs up" by one nucleotide, and a downstream stimulatory element, either an RNA pseudoknot or a very stable RNA stem-loop. These two elements are separated by six to eight nucleotides, a distance that places the 5' edge of the downstream stimulatory element in direct contact with the mRNA entry channel of the 30S ribosomal subunit. The precise mechanism by which the downstream RNA stimulates -1 PRF by the translocating ribosome remains unclear. This review summarizes the recent structural and biophysical studies of RNA pseudoknots and places this work in the context of our evolving mechanistic understanding of translation elongation. Support for the hypothesis that the downstream stimulatory element provides a kinetic barrier to the ribosome-mediated unfolding is discussed.
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Project description:The tomato Tm-1 gene confers resistance to tomato mosaic virus (ToMV). Here, we report that the extracts of Tm-1 tomato cells (GCR237) have properties that inhibit the in vitro RNA replication of WT ToMV more strongly than that of the Tm-1-resistance-breaking mutant of ToMV, LT1. We purified this inhibitory activity and identified a polypeptide of approximately 80 kDa (p80(GCR237)) using LC-tandem MS. The amino acid sequence of p80(GCR237) had no similarity to any characterized proteins. The p80(GCR237) gene cosegregated with Tm-1; transgenic expression of p80(GCR237) conferred resistance to ToMV within tomato plants; and the knockdown of p80(GCR237) sensitized Tm-1 tomato plants to ToMV, indicating that Tm-1 encodes p80(GCR237) itself. We further show that in vitro-synthesized Tm-1 (p80(GCR237)) protein binds to the replication proteins of WT ToMV and inhibits their function at a step before, but not after, the viral replication complex is formed on the membrane surfaces. Such binding was not observed for the replication proteins of LT1. These results suggest that Tm-1 (p80(GCR237)) inhibits the replication of WT ToMV RNA through binding to the replication proteins.
| S-EPMC1949341 | biostudies-literature
Project description:RNA can fold into a topological structure called a pseudoknot, composed of non-nested double-stranded stems connected by single-stranded loops. Our examination of the PseudoBase database of pseudoknotted RNA structures reveals asymmetries in the stem and loop lengths and provocative composition differences between the loops. By taking into account differences between major and minor grooves of the RNA double helix, we explain much of the asymmetry with a simple polymer physics model and statistical mechanical theory, with only one adjustable parameter.
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Project description:The available tools for conditional gene expression in Plasmodium falciparum are limited. Here, to enable reliable control of target gene expression, we build a system to efficiently modulate translation. We overcame several problems associated with other approaches for regulating gene expression in P. falciparum. Specifically, our system functions predictably across several native and engineered promoter contexts, and affords control over reporter and native parasite proteins irrespective of their subcellular compartmentalization. Induction and repression of gene expression are rapid, homogeneous and stable over prolonged periods. To demonstrate practical application of our system, we used it to reveal direct links between antimalarial drugs and their native parasite molecular target. This is an important outcome given the rapid spread of resistance, and intensified efforts to efficiently discover and optimize new antimalarial drugs. Overall, the studies presented highlight the utility of our system for broadly controlling gene expression and performing functional genetics in P. falciparum.
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