rRNA expansion segment 7 in eukaryotes: from Signature Fold to tentacles.

The ribosomal core is universally conserved across the tree of life. However, eukaryotic ribosomes contain diverse rRNA expansion segments (ESs) on their surfaces. Sites of ES insertions are predicted from sites of insertion of micro-ESs in archaea. Expansion segment 7 (ES7) is one of the most diverse regions of the ribosome, emanating from a short stem loop and ranging to over 750 nucleotides in mammals. We present secondary and full-atom 3D structures of ES7 from species spanning eukaryotic diversity. Our results are based on experimental 3D structures, the accretion model of ribosomal evolution, phylogenetic relationships, multiple sequence alignments, RNA folding algorithms and 3D modeling by RNAComposer. ES7 contains a distinct motif, the 'ES7 Signature Fold', which is generally invariant in 2D topology and 3D structure in all eukaryotic ribosomes. We establish a model in which ES7 developed over evolution through a series of elementary and recursive growth events. The data are sufficient to support an atomic-level accretion path for rRNA growth. The non-monophyletic distribution of some ES7 features across the phylogeny suggests acquisition via convergent processes. And finally, illustrating the power of our approach, we constructed the 2D and 3D structure of the entire LSU rRNA of Mus musculus.

Retroviral-like determinants and functions required for dimerization of Ty1 retrotransposon RNA.

During replication of long terminal repeat (LTR)-retrotransposons, their proteins and genome (g) RNA assemble into virus-like particles (VLPs) that are not infectious but functionally related to retroviral virions. Both virions and VLPs contain gRNA in a dimeric form, but contrary to retroviruses, little is known about how gRNA dimerization and packaging occurs in LTR-retrotransposons. The LTR-retrotransposon Ty1 from Saccharomyces cerevisiae is an informative model for studying LTR-retrotransposon and retrovirus replication. Using structural, mutational and functional analyses, we explored dimerization of Ty1 genomic RNA. We provide direct evidence that interactions of self-complementary PAL1 and PAL2 palindromic sequences localized within the 5'UTR are essential for Ty1 gRNA dimer formation. Mutations disrupting PAL1-PAL2 complementarity restricted RNA dimerization in vitro and Ty1 mobility in vivo. Although dimer formation and mobility of these mutants was inhibited, our work suggests that Ty1 RNA can dimerize via alternative contact points. In contrast to previous studies, we cannot confirm a role for PAL3, tRNAiMet as well as recently proposed initial kissing-loop interactions in dimer formation. Our data also supports the critical role of Ty1 Gag in RNA dimerization. Mature Ty1 Gag binds in the proximity of sequences involved in RNA dimerization and tRNAiMet annealing, but the 5' pseudoknot in Ty1 RNA may constitute a preferred Gag-binding site. Taken together, these results expand our understanding of genome dimerization and packaging strategies utilized by LTR-retroelements.

Eukaryotic Ribosomal Expansion Segments as Antimicrobial Targets.

Diversity in eukaryotic rRNA structure and function offers possibilities of therapeutic targets. Unlike ribosomes of prokaryotes, eukaryotic ribosomes contain species-specific rRNA expansion segments (ESs) with idiosyncratic structures and functions that are essential and specific to some organisms. Here we investigate expansion segment 7 (ES7), one of the largest and most variable expansions of the eukaryotic ribosome. We hypothesize that ES7 of the pathogenic fungi Candida albicans (ES7CA) could be a prototypic drug target. We show that isolated ES7CA folds reversibly to a native-like state. We developed a fluorescence displacement assay using an RNA binding fluorescent probe, F-neo. F-neo binds tightly to ES7CA with a Kd of 2.5 × 10-9 M but binds weakly to ES7 of humans (ES7HS) with a Kd estimated to be greater than 7 µM. The fluorescence displacement assay was used to investigate the affinities of a library of peptidic aminosugar conjugates (PAs) for ES7CA. For conjugates with highest affinities for ES7CA (NeoRH, NeoFH, and NeoYH), the lowest dose needed to induce mortality in C. albicans (minimum inhibitory concentration, MIC) was determined. PAs with the lowest MIC values were tested for cytotoxicity in HEK293T cells. Molecules with high affinity for ES7CA in vitro induce mortality in C. albicans but not in HEK293T cells. The results are consistent with the hypothesis that ESs represent useful targets for chemotherapeutics directed against eukaryotic pathogens.

A normal genetic variation modulates synaptic MMP-9 protein levels and the severity of schizophrenia symptoms.

Matrix metalloproteinase 9 (MMP-9) has recently emerged as a molecule that contributes to pathological synaptic plasticity in schizophrenia, but explanation of the underlying mechanisms has been missing. In the present study, we performed a phenotype-based genetic association study (PGAS) in > 1,000 schizophrenia patients from the Göttingen Research Association for Schizophrenia (GRAS) data collection and found an association between the MMP-9 rs20544 C/T single-nucleotide polymorphism (SNP) located in the 3'untranslated region (UTR) and the severity of a chronic delusional syndrome. In cultured neurons, the rs20544 SNP influenced synaptic MMP-9 activity and the morphology of dendritic spines. We demonstrated that Fragile X mental retardation protein (FMRP) bound the MMP-9 3'UTR We also found dramatic changes in RNA structure folding and alterations in the affinity of FMRP for MMP-9 RNA, depending on the SNP variant. Finally, we observed greater sensitivity to psychosis-related locomotor hyperactivity in Mmp-9 heterozygous mice. We propose a novel mechanism that involves MMP-9-dependent changes in dendritic spine morphology and the pathophysiology of schizophrenia, providing the first mechanistic insights into the way in which the single base change in the MMP-9 gene (rs20544) influences gene function and results in phenotypic changes observed in schizophrenia patients.

Structure of Ty1 Internally Initiated RNA Influences Restriction Factor Expression.

The long-terminal repeat retrotransposon Ty1 is the most abundant mobile genetic element in many Saccharomyces cerevisiae isolates. Ty1 retrotransposons contribute to the genetic diversity of host cells, but they can also act as an insertional mutagen and cause genetic instability. Interestingly, retrotransposition occurs at a low level despite a high level of Ty1 RNA, even though S. cerevisiae lacks the intrinsic defense mechanisms that other eukaryotes use to prevent transposon movement. p22 is a recently discovered Ty1 protein that inhibits retrotransposition in a dose-dependent manner. p22 is a truncated form of Gag encoded by internally initiated Ty1i RNA that contains two closely-spaced AUG codons. Mutations of either AUG codon compromise p22 translation. We found that both AUG codons were utilized and that translation efficiency depended on the Ty1i RNA structure. Structural features that stimulated p22 translation were context dependent and present only in Ty1i RNA. Destabilization of the 5' untranslated region (5' UTR) of Ty1i RNA decreased the p22 level, both in vitro and in vivo. Our data suggest that protein factors such as Gag could contribute to the stability and translational activity of Ty1i RNA through specific interactions with structural motifs in the RNA.

RNA-Puzzles Round III: 3D RNA structure prediction of five riboswitches and one ribozyme.

RNA-Puzzles is a collective experiment in blind 3D RNA structure prediction. We report here a third round of RNA-Puzzles. Five puzzles, 4, 8, 12, 13, 14, all structures of riboswitch aptamers and puzzle 7, a ribozyme structure, are included in this round of the experiment. The riboswitch structures include biological binding sites for small molecules (S-adenosyl methionine, cyclic diadenosine monophosphate, 5-amino 4-imidazole carboxamide riboside 5'-triphosphate, glutamine) and proteins (YbxF), and one set describes large conformational changes between ligand-free and ligand-bound states. The Varkud satellite ribozyme is the most recently solved structure of a known large ribozyme. All puzzles have established biological functions and require structural understanding to appreciate their molecular mechanisms. Through the use of fast-track experimental data, including multidimensional chemical mapping, and accurate prediction of RNA secondary structure, a large portion of the contacts in 3D have been predicted correctly leading to similar topologies for the top ranking predictions. Template-based and homology-derived predictions could predict structures to particularly high accuracies. However, achieving biological insights from de novo prediction of RNA 3D structures still depends on the size and complexity of the RNA. Blind computational predictions of RNA structures already appear to provide useful structural information in many cases. Similar to the previous RNA-Puzzles Round II experiment, the prediction of non-Watson-Crick interactions and the observed high atomic clash scores reveal a notable need for an algorithm of improvement. All prediction models and assessment results are available at http://ahsoka.u-strasbg.fr/rnapuzzles/.

Meeting report: Third Summer School on Innovative Approaches for Identification of Antiviral Agents (IAAASS).

The third Summer School on Innovative Approaches for Identification of Antiviral Agents (IAAASS) was held from September 28th to October 2nd, 2016 at the Sardegna Ricerche Research Park in Santa Margherita di Pula, Sardinia, Italy. The school brought together graduate students and postdoctoral fellows early in their careers with a faculty of internationally recognized experts, to encourage the sharing of knowledge and experience in virology research and drug development in an informal and interactive environment. The first IAAASS was held in Sardinia in 2012 and the second in 2014. The meetings provide a unique combination of plenary lectures on topics in virology, biochemistry, molecular modeling, crystallography and medicinal chemistry with small group sessions, in which students have the opportunity to ask questions and put forward their own ideas, and senior researchers offer advice, based on their own experience. This report summarizes presentations and presentations at the 3rd IAAASS.

Automated RNA 3D Structure Prediction with RNAComposer.

RNAs adopt specific structures to perform their activities and these are critical to virtually all RNA-mediated processes. Because of difficulties in experimentally assessing structures of large RNAs using NMR, X-ray crystallography, or cryo-microscopy, there is currently great demand for new high-resolution 3D structure prediction methods. Recently we reported on RNAComposer, a knowledge-based method for the fully automated RNA 3D structure prediction from a user-defined secondary structure. RNAComposer method is especially suited for structural biology users. Since our initial report in 2012, both servers, freely available at http://rnacomposer.ibch.poznan.pl and http://rnacomposer.cs.put.poznan.pl have been often visited. Therefore this chapter provides guidance for using RNAComposer and discusses points that should be considered when predicting 3D RNA structure. An application example presents current scope and limitations of RNAComposer.

Determinants of Genomic RNA Encapsidation in the Saccharomyces cerevisiae Long Terminal Repeat Retrotransposons Ty1 and Ty3.

Long-terminal repeat (LTR) retrotransposons are transposable genetic elements that replicate intracellularly, and can be considered progenitors of retroviruses. Ty1 and Ty3 are the most extensively characterized LTR retrotransposons whose RNA genomes provide the template for both protein translation and genomic RNA that is packaged into virus-like particles (VLPs) and reverse transcribed. Genomic RNAs are not divided into separate pools of translated and packaged RNAs, therefore their trafficking and packaging into VLPs requires an equilibrium between competing events. In this review, we focus on Ty1 and Ty3 genomic RNA trafficking and packaging as essential steps of retrotransposon propagation. We summarize the existing knowledge on genomic RNA sequences and structures essential to these processes, the role of Gag proteins in repression of genomic RNA translation, delivery to VLP assembly sites, and encapsidation.

Characterizing the functions of Ty1 Gag and the Gag-derived restriction factor p22/p18.

The long terminal repeat (LTR) and non-LTR retrotransposons comprise approximately half of the human genome, and we are only beginning to understand their influence on genome function and evolution. The LTR retrotransposon Ty1 is the most abundant mobile genetic element in the S. cerevisiae reference genome. Ty1 replicates via an RNA intermediate and shares several important structural and functional characteristics with retroviruses. However, unlike retroviruses Ty1 retrotransposition is not infectious. Retrotransposons integrations can cause mutations and genome instability. Despite the fact that S. cerevisiae lacks eukaryotic defense mechanisms such as RNAi, they maintain a relatively low copy number of the Ty1 retrotransposon in their genomes. A novel restriction factor derived from the C-terminal half of Gag (p22/p18) and encoded by internally initiated transcript inhibits retrotransposition in a dose-dependent manner. Therefore, Ty1 evolved a specific GAG organization and expression strategy to produce products both essential and antagonistic for retrotransposon movement. In this commentary we discuss our recent research aimed at defining steps of Ty1 replication influenced by p22/p18 with particular emphasis on the nucleic acid chaperone functions carried out by Gag and the restriction factor.

RNAComposer and RNA 3D structure prediction for nanotechnology.

RNAs adopt specific, stable tertiary architectures to perform their activities. Knowledge of RNA tertiary structure is fundamental to understand RNA functions beginning with transcription and ending with turnover. Contrary to advanced RNA secondary structure prediction algorithms, which allow good accuracy when experimental data are integrated into the prediction, tertiary structure prediction of large RNAs still remains a significant challenge. However, the field of RNA tertiary structure prediction is rapidly developing and new computational methods based on different strategies are emerging. RNAComposer is a user-friendly and freely available server for 3D structure prediction of RNA up to 500 nucleotide residues. RNAComposer employs fully automated fragment assembly based on RNA secondary structure specified by the user. Importantly, this method allows incorporation of distance restraints derived from the experimental data to strengthen the 3D predictions. The potential and limitations of RNAComposer are discussed and an application to RNA design for nanotechnology is presented.

The matrix domain contributes to the nucleic acid chaperone activity of HIV-2 Gag.

BACKGROUND: The Gag polyprotein is a multifunctional regulator of retroviral replication and major structural component of immature virions. The nucleic acid chaperone (NAC) activity is considered necessary to retroviral Gag functions, but so far, NAC activity has only been confirmed for HIV-1 and RSV Gag polyproteins. The nucleocapsid (NC) domain of Gag is proposed to be crucial for interactions with nucleic acids and NAC activity. The major function of matrix (MA) domain is targeting and binding of Gag to the plasma membrane but MA can also interact with RNA and influence NAC activity of Gag. Here, we characterize RNA binding properties and NAC activity of HIV-2 MA and Gag, lacking p6 domain (GagΔp6) and discuss potential contribution of NC and MA domains to HIV-2 GagΔp6 functions and interactions with RNA. RESULTS: We found that HIV-2 GagΔp6 is a robust nucleic acid chaperone. HIV-2 MA protein promotes nucleic acids aggregation and tRNA(Lys3) annealing in vitro. The NAC activity of HIV-2 NC is affected by salt which is in contrast to HIV-2 GagΔp6 and MA. At a physiological NaCl concentration the tRNA(Lys3) annealing activity of HIV-2 GagΔp6 or MA is higher than HIV-2 NC. The HIV-2 NC and GagΔp6 show strong binding to the packaging signal (Ψ) of HIV-2 RNA and preference for the purine-rich sequences, while MA protein binds mainly to G residues without favouring Ψ RNA. Moreover, HIV-2 GagΔp6 and NC promote HIV-2 RNA dimerization while our data do not support MA domain participation in this process in vitro. CONCLUSIONS: We present that contrary to HIV-1 MA, HIV-2 MA displays NAC activity and we propose that MA domain may enhance the activity of HIV-2 GagΔp6. The role of the MA domain in the NAC activity of Gag may differ significantly between HIV-1 and HIV-2. The HIV-2 NC and MA interactions with RNA are not equivalent. Even though both NC and MA can facilitate tRNA(Lys3) annealing, MA does not participate in RNA dimerization in vitro. Our data on HIV-2 indicate that the role of the MA domain in the NAC activity of Gag differs not only between, but also within, retroviral genera.

A self-encoded capsid derivative restricts Ty1 retrotransposition in Saccharomyces.

Retrotransposons and retroviral insertions have molded the genomes of many eukaryotes. Since retroelements transpose via an RNA intermediate, the additive nature of the replication cycle can result in massive increases in copy number if left unchecked. Host organisms have countered with several defense systems, including domestication of retroelement genes that now act as restriction factors to minimize propagation. We discovered a novel truncated form of the Saccharomyces Ty1 retrotransposon capsid protein, dubbed p22 that inhibits virus-like particle (VLP) assembly and function. The p22 restriction factor expands the repertoire of defense proteins targeting the capsid and highlights a novel host-parasite strategy. Instead of inhibiting all transposition by domesticating the restriction gene as a distinct locus, Ty1 and budding yeast may have coevolved a relationship that allows high levels of transposition when Ty1 copy numbers are low and progressively less transposition as copy numbers rise. Here, we offer a perspective on p22 restriction, including its mode of expression, effect on VLP functions, interactions with its target, properties as a nucleic acid chaperone, similarities to other restriction factors, and future directions.

The "complex" RNA post-transcriptional element of a "simple" retrovirus.

Replication of retroviruses and transposition of endogenous retroelements exploits a unique mechanism of post-transcriptional regulation as a means of exporting their incompletely-spliced mRNAs (which serve as both the genomic RNA and the template for protein synthesis). Following discovery of the Rev response element (RRE) that mediates nucleocytoplasmic export of the full-length and singly-spliced human immunodeficiency virus type 1 (HIV-1) genome, equivalent cis-acting regulatory elements have been characterized for both complex and simple retroviruses and retroelements, together with the obligate viral and host proteins with which they interact. The exception to this is the gammaretrovirus family of simple retroviruses, exemplified by reticuloendotheliosis virus (REV), murine leukemia virus (MLV) and xenotropic MLV-related retrovirus (XMRV). In this commentary, we discuss our recent data that reported structural and functional data on the MLV/XMRV post-transcriptional regulatory element (designated the PTE). The PTE was characterized by a highly-structured region of multiple stem-loops (SL1 - SL7) overlapping the pro and 5' portion of the pol open reading frames, comprising a bipartite export signal whose structures are separated by ∼1400 nt. In addition, structural probing suggested that SL3 nucleotides were involved in pseudoknot formation. These data, when compared with RNA transport elements of complex retroviruses (HIV) and simple murine retrotransposons (musD), collectively present an emerging picture that long-range tertiary interactions are critical mediators of their biological function.

Ty1 retrovirus-like element Gag contains overlapping restriction factor and nucleic acid chaperone functions.

Ty1 Gag comprises the capsid of virus-like particles and provides nucleic acid chaperone (NAC) functions during retrotransposition in budding yeast. A subgenomic Ty1 mRNA encodes a truncated Gag protein (p22) that is cleaved by Ty1 protease to form p18. p22/p18 strongly inhibits transposition and can be considered an element-encoded restriction factor. Here, we show that only p22 and its short derivatives restrict Ty1 mobility whereas other regions of GAG inhibit mobility weakly if at all. Mutational analyses suggest that p22/p18 is synthesized from either of two closely spaced AUG codons. Interestingly, AUG1p18 and AUG2p18 proteins display different properties, even though both contain a region crucial for RNA binding and NAC activity. AUG1p18 shows highly reduced NAC activity but specific binding to Ty1 RNA, whereas AUG2p18 shows the converse behavior. p22/p18 affects RNA encapsidation and a mutant derivative defective for RNA binding inhibits the RNA chaperone activity of the C-terminal region (CTR) of Gag-p45. Moreover, affinity pulldowns show that p18 and the CTR interact. These results support the idea that one aspect of Ty1 restriction involves inhibition of Gag-p45 NAC functions by p22/p18-Gag interactions.

RNA-Puzzles Round II: assessment of RNA structure prediction programs applied to three large RNA structures.

This paper is a report of a second round of RNA-Puzzles, a collective and blind experiment in three-dimensional (3D) RNA structure prediction. Three puzzles, Puzzles 5, 6, and 10, represented sequences of three large RNA structures with limited or no homology with previously solved RNA molecules. A lariat-capping ribozyme, as well as riboswitches complexed to adenosylcobalamin and tRNA, were predicted by seven groups using RNAComposer, ModeRNA/SimRNA, Vfold, Rosetta, DMD, MC-Fold, 3dRNA, and AMBER refinement. Some groups derived models using data from state-of-the-art chemical-mapping methods (SHAPE, DMS, CMCT, and mutate-and-map). The comparisons between the predictions and the three subsequently released crystallographic structures, solved at diffraction resolutions of 2.5-3.2 Å, were carried out automatically using various sets of quality indicators. The comparisons clearly demonstrate the state of present-day de novo prediction abilities as well as the limitations of these state-of-the-art methods. All of the best prediction models have similar topologies to the native structures, which suggests that computational methods for RNA structure prediction can already provide useful structural information for biological problems. However, the prediction accuracy for non-Watson-Crick interactions, key to proper folding of RNAs, is low and some predicted models had high Clash Scores. These two difficulties point to some of the continuing bottlenecks in RNA structure prediction. All submitted models are available for download at http://ahsoka.u-strasbg.fr/rnapuzzles/.

Gammaretrovirus mRNA expression is mediated by a novel, bipartite post-transcriptional regulatory element.

Post-transcriptional regulatory mechanisms of several complex and simple retroviruses and retroelements have been elucidated, with the exception of the gammaretrovirus family. We found that, similar to the other retroviruses, gag gene expression of MuLV and XMRV depends on post-transcriptional regulation mediated via an RNA sequence overlapping the pro-pol open reading frame, termed the Post-Transcriptional Element (PTE). PTE function can be replaced by heterologous RNA export elements, e.g. CTE of simian type D retroviruses. Alternatively, Gag particle production is achieved using an RNA/codon optimized gag gene. PTE function is transferable and can replace HIV Rev-RRE-regulated expression of HIV gag. Analysis of PTE by SHAPE revealed a highly structured RNA comprising seven stem-loop structures, with the 5' and 3' stem-loops forming an essential bipartite signal. MuLV and XMRV PTE share 98% identity and have highly similar RNA structures, with changes mostly located to single-stranded regions. PTE identification strongly suggests that all retroviruses and retroelements share common strategies of post-transcriptional gene regulation to produce Gag. Expression depends on complex RNA structures embedded within retroviral mRNA, in coding regions or the 3' untranslated region. These specific structures serve as recognition signals for either cellular or viral proteins.

Similarities and differences in the nucleic acid chaperone activity of HIV-2 and HIV-1 nucleocapsid proteins in vitro.

BACKGROUND: The nucleocapsid domain of Gag and mature nucleocapsid protein (NC) act as nucleic acid chaperones and facilitate folding of nucleic acids at critical steps of retroviral replication cycle. The basic N-terminus of HIV-1 NC protein was shown most important for the chaperone activity. The HIV-2 NC (NCp8) and HIV-1 NC (NCp7) proteins possess two highly conserved zinc fingers, flanked by basic residues. However, the NCp8 N-terminal domain is significantly shorter and contains less positively charged residues. This study characterizes previously unknown, nucleic acid chaperone activity of the HIV-2 NC protein. RESULTS: We have comparatively investigated the in vitro nucleic acid chaperone properties of the HIV-2 and HIV-1 NC proteins. Using substrates derived from the HIV-1 and HIV-2 genomes, we determined the ability of both proteins to chaperone nucleic acid aggregation, annealing and strand exchange in duplex structures. Both NC proteins displayed comparable, high annealing activity of HIV-1 TAR DNA and its complementary nucleic acid. Interesting differences between the two NC proteins were discovered when longer HIV substrates, particularly those derived from the HIV-2 genome, were used in chaperone assays. In contrast to NCp7, NCp8 weakly facilitates annealing of HIV-2 TAR RNA to its complementary TAR (-) DNA. NCp8 is also unable to efficiently stimulate tRNALys3 annealing to its respective HIV-2 PBS motif. Using truncated NCp8 peptide, we demonstrated that despite the fact that the N-terminus of NCp8 differs from that of NCp7, this domain is essential for NCp8 activity. CONCLUSION: Our data demonstrate that the HIV-2 NC protein displays reduced nucleic acid chaperone activity compared to that of HIV-1 NC. We found that NCp8 activity is limited by substrate length and stability to a greater degree than that of NCp7. This is especially interesting in light of the fact that the HIV-2 5'UTR is more structured than that of HIV-1. The reduced chaperone activity observed with NCp8 may influence the efficiency of reverse transcription and other key steps of the HIV-2 replication cycle.

Influence of RNA structural elements on Ty1 retrotransposition.

The long-terminal repeat (LTR)-retrotransposon Ty1 is a mobile genetic element that replicates through an RNA intermediate. Retroelement genomic transcripts contain internal structures fundamental to gene expression and propagation. In addition, long non-coding antisense RNAs overlap the 5'-terminal region of the genomic RNA and confer post-translational copy number control. Although LTR- retrotransposons are functionally related to retroviruses, little is known about the structural determinants required for genomic RNA packaging or reverse transcription. This commentary summarizes two recent papers that provide the first snapshot of genomic RNA structures from the retrotransposon Ty1 involved in transposition. We combined structural approaches with functional and genetic assays to determine if antisense RNAs anneal with the genomic RNA. Analysis of various steps in the Ty1 life cycle showed that a novel RNA pseudoknot contributes to retrotransposon function. Comparing different RNA states provides additional information about regions potentially involved in Ty1 RNA dimerization or packaging.

RNA secondary structure prediction using high-throughput SHAPE.

Understanding the function of RNA involved in biological processes requires a thorough knowledge of RNA structure. Toward this end, the methodology dubbed "high-throughput selective 2' hydroxyl acylation analyzed by primer extension", or SHAPE, allows prediction of RNA secondary structure with single nucleotide resolution. This approach utilizes chemical probing agents that preferentially acylate single stranded or flexible regions of RNA in aqueous solution. Sites of chemical modification are detected by reverse transcription of the modified RNA, and the products of this reaction are fractionated by automated capillary electrophoresis (CE). Since reverse transcriptase pauses at those RNA nucleotides modified by the SHAPE reagents, the resulting cDNA library indirectly maps those ribonucleotides that are single stranded in the context of the folded RNA. Using ShapeFinder software, the electropherograms produced by automated CE are processed and converted into nucleotide reactivity tables that are themselves converted into pseudo-energy constraints used in the RNAStructure (v5.3) prediction algorithm. The two-dimensional RNA structures obtained by combining SHAPE probing with in silico RNA secondary structure prediction have been found to be far more accurate than structures obtained using either method alone.