RNASTAR: an RNA STructural Alignment Repository that provides insight into the evolution of natural and artificial RNAs.

Automated RNA alignment algorithms often fail to recapture the essential conserved sites that are critical for function. To assist in the refinement of these algorithms, we manually curated a set of 148 alignments with a total of 9600 unique sequences, in which each alignment was backed by at least one crystal or NMR structure. These alignments included both naturally and artificially selected molecules. We used principles of isostericity to improve the alignments from an average of 83%-94% isosteric base pairs. We expect that this alignment collection will assist in a wide range of benchmarking efforts and provide new insight into evolutionary principles governing change in RNA structural motifs. The improved alignments have been contributed to the Rfam database.

Simple, recurring RNA binding sites for L-arginine.

Seven new arginine binding motifs have been selected from a heterogeneous RNA pool containing 17, 25, and 50mer randomized tracts, yielding 131 independently derived binding sites that are multiply isolated. The shortest 17mer random region is sufficient to build varied arginine binding sites using five different conserved motifs (motifs 1a, 1b, 1c, 2, and 4). Dissociation constants are in the fractional millimolar to millimolar range. Binding sites are amino acid side-chain specific and discriminate moderately between L- and D-stereoisomers of arginine, suggesting a molecular focus on side-chain guanidinium. An arginine coding triplet (codon/anticodon) is highly conserved within the largest family of Arg sites (72% of all sequences), as has also been found in minimal, most prevalent RNA binding sites for Ile, His, and Trp.

Stable tRNA-based phylogenies using only 76 nucleotides.

tRNAs are among the most ancient, highly conserved sequences on earth, but are often thought to be poor phylogenetic markers because they are short, often subject to horizontal gene transfer, and easily change specificity. Here we use an algorithm now commonly used in microbial ecology, UniFrac, to cluster 175 genomes spanning all three domains of life based on the phylogenetic relationships among their complete tRNA pools. We find that the overall pattern of similarities and differences in the tRNA pools recaptures universal phylogeny to a remarkable extent, and that the resulting tree is similar to the distribution of bootstrapped rRNA trees from the same genomes. In contrast, the trees derived from tRNAs of identical specificity or of individual isoacceptors generally produced trees of lower quality. However, some tRNA isoacceptors were very good predictors of the overall pattern of organismal evolution. These results show that UniFrac can extract meaningful biological patterns from even phylogenies with high level of statistical inaccuracy and horizontal gene transfer, and that, overall, the pattern of tRNA evolution tracks universal phylogeny and provides a background against which we can test hypotheses about the evolution of individual isoacceptors.

Nucleotides that are essential but not conserved; a sufficient L-tryptophan site in RNA.

Conservation is often used to define essential sequences within RNA sites. However, conservation finds only invariant sequence elements that are necessary for function, rather than finding a set of sequence elements sufficient for function. Biochemical studies in several systems-including the hammerhead ribozyme and the purine riboswitch-find additional elements, such as loop-loop interactions, required for function yet not phylogenetically conserved. Here we define a critical test of sufficiency: We embed a minimal, apparently sufficient motif for binding the amino acid tryptophan in a random-sequence background and ask whether we obtain functional molecules. After a negative result, we use a combination of three-dimensional structural modeling, selection, designed mutations, high-throughput sequencing, and bioinformatics to explore functional insufficiency. This reveals an essential unpaired G in a diverse structural context, varied sequence, and flexible distance from the invariant internal loop binding site identified previously. Addition of the new element yields a sufficient binding site by the insertion criterion, binding tryptophan in 22 out of 23 tries. Random insertion testing for site sufficiency seems likely to be broadly revealing.

Boulder ALignment Editor (ALE): a web-based RNA alignment tool.

SUMMARY: The explosion of interest in non-coding RNAs, together with improvements in RNA X-ray crystallography, has led to a rapid increase in RNA structures at atomic resolution from 847 in 2005 to 1900 in 2010. The success of whole-genome sequencing has led to an explosive growth of unaligned homologous sequences. Consequently, there is a compelling and urgent need for user-friendly tools for producing structure-informed RNA alignments. Most alignment software considers the primary sequence alone; some specialized alignment software can also include Watson-Crick base pairs, but none adequately addresses the needs introduced by the rapid influx of both sequence and structural data. Therefore, we have developed the Boulder ALignment Editor (ALE), which is a web-based RNA alignment editor, designed for editing and assessing alignments using structural information. Some features of BoulderALE include the annotation and evaluation of an alignment based on isostericity of Watson-Crick and non-Watson-Crick base pairs, along with the collapsing (horizontally and vertically) of the alignment, while maintaining the ability to edit the alignment. AVAILABILITY: http://www.microbio.me/boulderale.

RNA-amino acid binding: a stereochemical era for the genetic code.

By combining crystallographic and NMR structural data for RNA-bound amino acids within riboswitches, aptamers, and RNPs, chemical principles governing specific RNA interaction with amino acids can be deduced. Such principles, which we summarize in a "polar profile", are useful in explaining newly selected specific RNA binding sites for free amino acids bearing varied side chains charged, neutral polar, aliphatic, and aromatic. Such amino acid sites can be queried for parallels to the genetic code. Using recent sequences for 337 independent binding sites directed to 8 amino acids and containing 18,551 nucleotides in all, we show a highly robust connection between amino acids and cognate coding triplets within their RNA binding sites. The apparent probability (P) that cognate triplets around these sites are unrelated to binding sites is congruent with 5.3 x 10(-45) for codons overall, and P congruent with 2.1 x 10(-46) for cognate anticodons. Therefore, some triplets are unequivocally localized near their present amino acids. Accordingly, there was likely a stereochemical era during evolution of the genetic code, relying on chemical interactions between amino acids and the tertiary structures of RNA binding sites. Use of cognate coding triplets in RNA binding sites is nevertheless sparse, with only 21% of possible triplets appearing. Reasoning from such broad recurrent trends in our results, a majority (approximately 75%) of modern amino acids entered the code in this stereochemical era; nevertheless, a minority (approximately 21%) of modern codons and anticodons were assigned via RNA binding sites. A Direct RNA Template scheme embodying a credible early history for coded peptide synthesis is readily constructed based on these observations.

Nucleotides adjacent to the ligand-binding pocket are linked to activity tuning in the purine riboswitch.

Direct sensing of intracellular metabolite concentrations by riboswitch RNAs provides an economical and rapid means to maintain metabolic homeostasis. Since many organisms employ the same class of riboswitch to control different genes or transcription units, it is likely that functional variation exists in riboswitches such that activity is tuned to meet cellular needs. Using a bioinformatic approach, we have identified a region of the purine riboswitch aptamer domain that displays conservation patterns linked to riboswitch activity. Aptamer domain compositions within this region can be divided into nine classes that display a spectrum of activities. Naturally occurring compositions in this region favor rapid association rate constants and slow dissociation rate constants for ligand binding. Using X-ray crystallography and chemical probing, we demonstrate that both the free and bound states are influenced by the composition of this region and that modest sequence alterations have a dramatic impact on activity. The introduction of non-natural compositions result in the inability to regulate gene expression in vivo, suggesting that aptamer domain activity is highly plastic and thus readily tunable to meet cellular needs.

Human Immunodeficiency Virus nef signature sequences are associated with pulmonary hypertension.

Severe pulmonary hypertension (PH) associated with vascular remodeling is a long-term complication of HIV infection (HIV-PH) affecting 1/200 infected individuals vs. 1/200,000 frequency in the uninfected population. Factors accounting for increased PH susceptibility in HIV-infected individuals are unknown. Rhesus macaques infected with chimeric SHIVnef virions but not with SIV display PH-like pulmonary vascular remodeling suggesting that HIV-Nef is associated with PH; these monkeys showed changes in nef sequences that correlated with pathogenesis after passage in vivo. We further examined whether HIV-nef alleles in HIV-PH subjects have signature sequences associated with the disease phenotype. We evaluated specimens from participants with and without HIV-PH from European Registries and validated results with samples collected as part of the Lung-HIV Studies in San Francisco. We found that 10 polymorphisms in nef were overrepresented in blood cells or lung tissue specimens from European HIV-PH individuals but significantly less frequent in HIV-infected individuals without PH. These polymorphisms mapped to known functional domains in Nef. In the validation cohort, 7/10 polymorphisms in the HIV-nef gene were confirmed; these polymorphisms arose independently from viral load, CD4(+) T cell counts, length of infection, and antiretroviral therapy status. Two out of 10 polymorphisms were previously reported in macaques with PH-like pulmonary vascular remodeling. Cloned recombinant Nef proteins from clinical samples down-regulated CD4, suggesting that these primary isolates are functional. This study offers new insights into the association between Nef polymorphisms in functional domains and the HIV-PH phenotype. The utility of these polymorphisms as predictors of PH should be examined in a larger population.

MotifCluster: an interactive online tool for clustering and visualizing sequences using shared motifs.

MotifCluster finds related motifs in a set of sequences, and clusters the sequences into families using the motifs they contain. MotifCluster, at http://bmf.colorado.edu/motifcluster, lets users test whether proteins are related, cluster sequences by shared conserved motifs, and visualize motifs mapped onto trees, sequences and three-dimensional structures. We demonstrate MotifCluster's accuracy using gold-standard protein superfamilies; using recommended settings, families were assigned to the correct superfamilies with 0.17% false positive and no false negative assignments.

PyCogent: a toolkit for making sense from sequence.

We have implemented in Python the COmparative GENomic Toolkit, a fully integrated and thoroughly tested framework for novel probabilistic analyses of biological sequences, devising workflows, and generating publication quality graphics. PyCogent includes connectors to remote databases, built-in generalized probabilistic techniques for working with biological sequences, and controllers for third-party applications. The toolkit takes advantage of parallel architectures and runs on a range of hardware and operating systems, and is available under the general public license from http://sourceforge.net/projects/pycogent.

DivergentSet, a tool for picking non-redundant sequences from large sequence collections.

DivergentSet addresses the important but so far neglected bioinformatics task of choosing a representative set of sequences from a larger collection. We found that using a phylogenetic tree to guide the construction of divergent sets of sequences can be up to 2 orders of magnitude faster than the naive method of using a full distance matrix. By providing a user-friendly interface (available online) that integrates the tasks of finding additional sequences, building and refining the divergent set, producing random divergent sets from the same sequences, and exporting identifiers, this software facilitates a wide range of bioinformatics analyses including finding significant motifs and covariations. As an example application of DivergentSet, we demonstrate that the motifs identified by the motif-finding package MEME (Motif Elicitation by Maximum Entropy) are highly unstable with respect to the specific choice of sequences. This instability suggests that the types of sensitivity analysis enabled by DivergentSet may be widely useful for identifying the motifs of biological significance.

tRNA creation by hairpin duplication.

Many studies have suggested that the modern cloverleaf structure of tRNA may have arisen through duplication of a primordial hairpin, but the timing of this duplication event has been unclear. Here we measure the level of sequence identity between the two halves of each of a large sample of tRNAs and compare this level to that of chimeric tRNAs constructed either within or between groups defined by phylogeny and/or specificity. We find that actual tRNAs have significantly more matches between the two halves than do random sequences that can form the tRNA structure, but there is no difference in the average level of matching between the two halves of an individual tRNA and the average level of matching between the two halves of the chimeric tRNAs in any of the sets we constructed. These results support the hypothesis that the modern tRNA cloverleaf arose from a single hairpin duplication prior to the divergence of modern tRNA specificities and the three domains of life.