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From Structure Prediction to Genomic Screens for Novel Non-Coding RNAs
Non-coding RNAs (ncRNAs) are receiving more and more attention not only as an abundant class of genes, but also as regulatory structural elements (some located in mRNAs). A key feature of RNA function is its structure. Computational methods were developed early for folding and prediction of RNA structure with the aim of assisting in functional analysis. With the discovery of more and more ncRNAs, it has become clear that a large fraction of these are highly structured. Interestingly, a large part of the structure is comprised of regular Watson-Crick and GU wobble base pairs. This and the increased amount of available genomes have made it possible to employ structure-based methods for genomic screens. The field has moved from folding prediction of single sequences to computational screens for ncRNAs in genomic sequence using the RNA structure as the main characteristic feature. Whereas early methods focused on energy-directed folding of single sequences, comparative analysis based on structure preserving changes of base pairs has been efficient in improving accuracy, and today this constitutes a key component in genomic screens. Here, we cover the basic principles of RNA folding and touch upon some of the concepts in current methods that have been applied in genomic screens for de novo RNA structures in searches for novel ncRNA genes and regulatory RNA structure on mRNAs. We discuss the strengths and weaknesses of the different strategies and how they can complement each other
Chance and Necessity in Evolution: Lessons from RNA
The relationship between sequences and secondary structures or shapes in RNA
exhibits robust statistical properties summarized by three notions: (1) the
notion of a typical shape (that among all sequences of fixed length certain
shapes are realized much more frequently than others), (2) the notion of shape
space covering (that all typical shapes are realized in a small neighborhood of
any random sequence), and (3) the notion of a neutral network (that sequences
folding into the same typical shape form networks that percolate through
sequence space). Neutral networks loosen the requirements on the mutation rate
for selection to remain effective. The original (genotypic) error threshold has
to be reformulated in terms of a phenotypic error threshold. With regard to
adaptation, neutrality has two seemingly contradictory effects: It acts as a
buffer against mutations ensuring that a phenotype is preserved. Yet it is
deeply enabling, because it permits evolutionary change to occur by allowing
the sequence context to vary silently until a single point mutation can become
phenotypically consequential. Neutrality also influences predictability of
adaptive trajectories in seemingly contradictory ways. On the one hand it
increases the uncertainty of their genotypic trace. At the same time neutrality
structures the access from one shape to another, thereby inducing a topology
among RNA shapes which permits a distinction between continuous and
discontinuous shape transformations. To the extent that adaptive trajectories
must undergo such transformations, their phenotypic trace becomes more
predictable.Comment: 37 pages, 14 figures; 1998 CNLS conference; high quality figures at
http://www.santafe.edu/~walte
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PATTERNA: transcriptome-wide search for functional RNA elements via structural data signatures.
Establishing a link between RNA structure and function remains a great challenge in RNA biology. The emergence of high-throughput structure profiling experiments is revolutionizing our ability to decipher structure, yet principled approaches for extracting information on structural elements directly from these data sets are lacking. We present PATTERNA, an unsupervised pattern recognition algorithm that rapidly mines RNA structure motifs from profiling data. We demonstrate that PATTERNA detects motifs with an accuracy comparable to commonly used thermodynamic models and highlight its utility in automating data-directed structure modeling from large data sets. PATTERNA is versatile and compatible with diverse profiling techniques and experimental conditions
A complex adaptive systems approach to the kinetic folding of RNA
The kinetic folding of RNA sequences into secondary structures is modeled as
a complex adaptive system, the components of which are possible RNA structural
rearrangements (SRs) and their associated bases and base pairs. RNA bases and
base pairs engage in local stacking interactions that determine the
probabilities (or fitnesses) of possible SRs. Meanwhile, selection operates at
the level of SRs; an autonomous stochastic process periodically (i.e., from one
time step to another) selects a subset of possible SRs for realization based on
the fitnesses of the SRs. Using examples based on selected natural and
synthetic RNAs, the model is shown to qualitatively reproduce characteristic
(nonlinear) RNA folding dynamics such as the attainment by RNAs of alternative
stable states. Possible applications of the model to the analysis of properties
of fitness landscapes, and of the RNA sequence to structure mapping are
discussed.Comment: 23 pages, 4 figures, 2 tables, to be published in BioSystems (Note:
updated 2 references
Understanding the errors of SHAPE-directed RNA structure modeling
Single-nucleotide-resolution chemical mapping for structured RNA is being
rapidly advanced by new chemistries, faster readouts, and coupling to
computational algorithms. Recent tests have shown that selective 2'-hydroxyl
acylation by primer extension (SHAPE) can give near-zero error rates (0-2%) in
modeling the helices of RNA secondary structure. Here, we benchmark the method
using six molecules for which crystallographic data are available: tRNA(phe)
and 5S rRNA from Escherichia coli, the P4-P6 domain of the Tetrahymena group I
ribozyme, and ligand-bound domains from riboswitches for adenine, cyclic
di-GMP, and glycine. SHAPE-directed modeling of these highly structured RNAs
gave an overall false negative rate (FNR) of 17% and a false discovery rate
(FDR) of 21%, with at least one helix prediction error in five of the six
cases. Extensive variations of data processing, normalization, and modeling
parameters did not significantly mitigate modeling errors. Only one varation,
filtering out data collected with deoxyinosine triphosphate during primer
extension, gave a modest improvement (FNR = 12%, and FDR = 14%). The residual
structure modeling errors are explained by the insufficient information content
of these RNAs' SHAPE data, as evaluated by a nonparametric bootstrapping
analysis. Beyond these benchmark cases, bootstrapping suggests a low level of
confidence (<50%) in the majority of helices in a previously proposed
SHAPE-directed model for the HIV-1 RNA genome. Thus, SHAPE-directed RNA
modeling is not always unambiguous, and helix-by-helix confidence estimates, as
described herein, may be critical for interpreting results from this powerful
methodology.Comment: Biochemistry, Article ASAP (Aug. 15, 2011
Replication enhancer elements within the open reading frame of tick-borne encephalitis virus and their evolution within the Flavivirus genus
We provide experimental evidence of a replication enhancer element (REE) within the capsid gene of tick-borne encephalitis virus (TBEV, genus Flavivirus). Thermodynamic and phylogenetic analyses predicted that the REE folds as a long stable stem–loop (designated SL6), conserved among all tick-borne flaviviruses (TBFV). Homologous sequences and potential base pairing were found in the corresponding regions of mosquito-borne flaviviruses, but not in more genetically distant flaviviruses. To investigate the role
of SL6, nucleotide substitutions were introduced which changed a conserved hexanucleotide motif, the conformation of the terminal loop and the base-paired dsRNA stacking. Substitutions were made within a TBEV reverse genetic system and recovered mutants were compared for plaque
morphology, single-step replication kinetics and cytopathic effect. The greatest phenotypic changes were observed in mutants with a destabilized stem. Point mutations in the conserved hexanucleotide motif of the terminal loop caused
moderate virus attenuation. However, all mutants
eventually reached the titre of wild-type virus late post-infection. Thus, although not essential for growth in tissue culture, the SL6 REE acts to up-regulate virus replication. We hypothesize that this modulatory role may be important for TBEV survival in nature, where the virus circulates by non-viraemic transmission between infected and
non-infected ticks, during co-feeding on local rodents
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