Correlating SHAPE signatures with three-dimensional RNA structures

Selective 2'-hydroxyl acylation analyzed by primer extension (SHAPE) is a facile technique for quantitative analysis of RNA secondary structure. In general, low SHAPE signal values indicate Watson-Crick base-pairing, and high values indicate positions that are single-stranded within the RNA structure. However, the relationship of SHAPE signals to structural properties such as non-Watson-Crick base-pairing or stacking has thus far not been thoroughly investigated. Here, we present results of SHAPE experiments performed on several RNAs with published three-dimensional structures. This strategy allows us to analyze the results in terms of correlations between chemical reactivities and structural properties of the respective nucleotide, such as different types of base-pairing, stacking, and phosphate-backbone interactions. We find that the RNA SHAPE signal is strongly correlated with cis-Watson-Crick/Watson-Crick base-pairing and is to a remarkable degree not dependent on other structural properties with the exception of stacking. We subsequently generated probabilistic models that estimate the likelihood that a residue with a given SHAPE score participates in base-pairing. We show that several models that take SHAPE scores of adjacent residues into account perform better in predicting base-pairing compared with individual SHAPE scores. This underscores the context sensitivity of SHAPE and provides a framework for an improved interpretation of the response of RNA to chemical modification.     

Characteristic chemical probing patterns of loop motifs improve prediction accuracy of RNA secondary structures

RNA structures play a fundamental role in nearly every aspect of cellular physiology and pathology. Gaining insights into the functions of RNA molecules requires accurate predictions of RNA secondary structures. However, the existing thermodynamic folding models remain less accurate than desired, even when chemical probing data, such as selective 2′-hydroxyl acylation analyzed by primer extension (SHAPE) reactivities, are used as restraints. Unlike most SHAPE-directed algorithms that only consider SHAPE restraints for base pairing, we extract two-dimensional structural features encoded in SHAPE data and establish robust relationships between characteristic SHAPE patterns and loop motifs of various types (hairpin, internal, and bulge) and lengths (2–11 nucleotides). Such characteristic SHAPE patterns are closely related to the sugar pucker conformations of loop residues. Based on these patterns, we propose a computational method, SHAPELoop, which refines the predicted results of the existing methods, thereby further improving their prediction accuracy. In addition, SHAPELoop can provide information about local or global structural rearrangements (including pseudoknots) and help researchers to easily test their hypothesized secondary structures.     

RNA secondary structure modeling at consistent high accuracy using differential SHAPE

RNA secondary structure modeling is a challenging problem, and recent successes have raised the standards for accuracy, consistency, and tractability. Large increases in accuracy have been achieved by including data on reactivity toward chemical probes: Incorporation of 1M7 SHAPE reactivity data into an mfold-class algorithm results in median accuracies for base pair prediction that exceed 90%. However, a few RNA structures are modeled with significantly lower accuracy. Here, we show that incorporating differential reactivities from the NMIA and 1M6 reagents—which detect noncanonical and tertiary interactions—into prediction algorithms results in highly accurate secondary structure models for RNAs that were previously shown to be difficult to model. For these RNAs, 93% of accepted canonical base pairs were recovered in SHAPE-directed models. Discrepancies between accepted and modeled structures were small and appear to reflect genuine structural differences. Three-reagent SHAPE-directed modeling scales concisely to structurally complex RNAs to resolve the in-solution secondary structure analysis problem for many classes of RNA.     

Synergistic SHAPE/Single-Molecule Deconvolution of RNA Conformation under Physiological Conditions

Structural RNA domains are widely involved in the regulation of biological functions, such as gene expression, gene modification, and gene repair. Activity of these dynamic regions depends sensitively on the global fold of the RNA, in particular, on the binding affinity of individual conformations to effector molecules in solution. Consequently, both the 1) structure and 2) conformational dynamics of noncoding RNAs prove to be essential in understanding the coupling that results in biological function. Toward this end, we recently reported observation of three conformational states in the metal-induced folding pathway of the tRNA-like structure domain of Brome Mosaic Virus, via single-molecule fluorescence resonance energy transfer studies. We report herein selective 2'-hydroxyl acylation analyzed by primer extension (SHAPE)-directed structure predictions as a function of metal ion concentrations ([Mⁿ⁺]) to confirm the three-state folding model, as well as test 2° structure models from the literature. Specifically, SHAPE reactivity data mapped onto literature models agrees well with the secondary structures observed at 0–10 mM [Mg²⁺], with only minor discrepancies in the E hairpin domain at low [Mg²⁺]. SHAPE probing and SHAPE-directed structure predictions further confirm the stepwise unfolding pathway previously observed in our single-molecule studies. Of special relevance, this means that reduction in the metal-ion concentration unfolds the 3′ pseudoknot interaction before unfolding the long-range stem interaction. This work highlights the synergistic power of combining 1) single-molecule Förster resonance energy transfer and 2) SHAPE-directed structure-probing studies for detailed analysis of multiple RNA conformational states. In particular, single-molecule guided deconvolution of the SHAPE reactivities permits 2° structure predictions of isolated RNA conformations, thereby substantially improving on traditional limitations associated with current structure prediction algorithms.     

Rotavirus replication: plus-sense templates for double-stranded RNA synthesis are made in viroplasms

Rotavirus plus-strand RNAs not only direct protein synthesis but also serve as templates for the synthesis of the segmented double-stranded RNA (dsRNA) genome. In this study, we identified short-interfering RNAs (siRNAs) for viral genes 5, 8, and 9 that suppressed the expression of NSP1, a nonessential protein; NSP2, a component of viral replication factories (viroplasms); and VP7, an outer capsid protein, respectively. The loss of NSP2 expression inhibited viroplasm formation, genome replication, virion assembly, and synthesis of the other viral proteins. In contrast, the loss of VP7 expression had no effect on genome replication; instead, it inhibited only outer-capsid morphogenesis. Similarly, neither genome replication nor any other event of the viral life cycle was affected by the loss of NSP1. The data indicate that plus-strand RNAs templating dsRNA synthesis within viroplasms are not susceptible to siRNA-induced RNase degradation. In contrast, plus-strand RNAs templating protein synthesis in the cytosol are susceptible to degradation and thus are not the likely source of plus-strand RNAs for dsRNA synthesis in viroplasms. Indeed, immunofluorescence analysis of bromouridine (BrU)-labeled RNA made in infected cells provided evidence that plus-strand RNAs are synthesized within viroplasms. Furthermore, transfection of BrU-labeled viral plus-strand RNA into infected cells suggested that plus-strand RNAs introduced into the cytosol do not localize to viroplasms. From these results, we propose that plus-strand RNAs synthesized within viroplasms are the primary source of templates for genome replication and that trafficking pathways do not exist within the cytosol that transport plus-strand RNAs to viroplasms. The lack of such pathways confounds the development of reverse genetics systems for rotavirus.     

Crystallographic Analysis of Rotavirus NSP2-RNA Complex Reveals Specific Recognition of 5' GG Sequence for RTPase Activity

Rotavirus nonstructural protein NSP2, a functional octamer, is critical for the formation of viroplasms, which are exclusive sites for replication and packaging of the segmented double-stranded RNA (dsRNA) rotavirus genome. As a component of replication intermediates, NSP2 is also implicated in various replication-related activities. In addition to sequence-independent single-stranded RNA-binding and helix-destabilizing activities, NSP2 exhibits monomer-associated nucleoside and 5' RNA triphosphatase (NTPase/RTPase) activities that are mediated by a conserved H225 residue within a narrow enzymatic cleft. Lack of a 5' γ-phosphate is a common feature of the negative-strand RNA [(-)RNA] of the packaged dsRNA segments in rotavirus. Strikingly, all (-)RNAs (of group A rotaviruses) have a 5' GG dinucleotide sequence. As the only rotavirus protein with 5' RTPase activity, NSP2 is implicated in the removal of the γ-phosphate from the rotavirus (-)RNA. To understand how NSP2, despite its sequence-independent RNA-binding property, recognizes (-)RNA to hydrolyze the γ-phosphate within the catalytic cleft, we determined a crystal structure of NSP2 in complex with the 5' consensus sequence of minus-strand rotavirus RNA. Our studies show that the 5' GG of the bound oligoribonucleotide interacts extensively with highly conserved residues in the NSP2 enzymatic cleft. Although these residues provide GG-specific interactions, surface plasmon resonance studies suggest that the C-terminal helix and other basic residues outside the enzymatic cleft account for sequence-independent RNA binding of NSP2. A novel observation from our studies, which may have implications in viroplasm formation, is that the C-terminal helix of NSP2 exhibits two distinct conformations and engages in domain-swapping interactions, which result in the formation of NSP2 octamer chains.     

A novel SHAPE reagent enables the analysis of RNA structure in living cells with unprecedented accuracy

Due to the mounting evidence that RNA structure plays a critical role in regulating almost any physiological as well as pathological process, being able to accurately define the folding of RNA molecules within living cells has become a crucial need. We introduce here 2-aminopyridine-3-carboxylic acid imidazolide (2A3), as a general probe for the interrogation of RNA structures in vivo. 2A3 shows moderate improvements with respect to the state-of-the-art selective 2′-hydroxyl acylation analyzed by primer extension (SHAPE) reagent NAI on naked RNA under in vitro conditions, but it significantly outperforms NAI when probing RNA structure in vivo, particularly in bacteria, underlining its increased ability to permeate biological membranes. When used as a restraint to drive RNA structure prediction, data derived by SHAPE-MaP with 2A3 yields more accurate predictions than NAI-derived data. Due to its extreme efficiency and accuracy, we can anticipate that 2A3 will rapidly take over conventional SHAPE reagents for probing RNA structures both in vitro and in vivo.     

RNA-binding activity of the rotavirus phosphoprotein NSP5 includes affinity for double-stranded RNA

Phosphoprotein NSP5 is a component of replication intermediates that catalyze the synthesis of the segmented double-stranded RNA (dsRNA) rotavirus genome. To study the role of the protein in viral replication, His-tagged NSP5 was expressed in bacteria and purified by affinity chromatography. In vitro phosphorylation assays showed that NSP5 alone contains minimal autokinase activity but undergoes hyperphosphorylation when combined with the NTPase and helix-destabilizing protein NSP2. Hence, NSP2 mediates the hyperphosphorylation of NSP5 in the absence of other viral or cellular proteins. RNA-binding assays demonstrated that NSP5 has unique nonspecific RNA-binding activity, recognizing single-stranded RNA and dsRNA with similar affinities. The possible functions of the RNA-binding activities of NSP5 are to cooperate with NSP2 in the destabilization of RNA secondary structures and in the packaging of RNA and/or to prevent the interferon-induced dsRNA-dependent activation of the protein kinase PKR.     

Residues of the rotavirus RNA-dependent RNA polymerase template entry tunnel that mediate RNA recognition and genome replication

To replicate its segmented, double-stranded RNA (dsRNA) genome, the rotavirus RNA-dependent RNA polymerase, VP1, must recognize viral plus-strand RNAs (+RNAs) and guide them into the catalytic center. VP1 binds to the conserved 3' end of rotavirus +RNAs via both sequence-dependent and sequence-independent contacts. Sequence-dependent contacts permit recognition of viral +RNAs and specify an autoinhibited positioning of the template within the catalytic site. However, the contributions to dsRNA synthesis of sequence-dependent and sequence-independent VP1-RNA interactions remain unclear. To analyze the importance of VP1 residues that interact with +RNA on genome replication, we engineered mutant VP1 proteins and assayed their capacity to synthesize dsRNA in vitro. Our results showed that, individually, mutation of residues that interact specifically with RNA bases did not diminish replication levels. However, simultaneous mutations led to significantly lower levels of dsRNA product, presumably due to impaired recruitment of +RNA templates. In contrast, point mutations of sequence-independent RNA contact residues led to severely diminished replication, likely as a result of improper positioning of templates at the catalytic site. A noteworthy exception was a K419A mutation that enhanced the initiation capacity and product elongation rate of VP1. The specific chemistry of Lys419 and its position at a narrow region of the template entry tunnel appear to contribute to its capacity to moderate replication. Together, our findings suggest that distinct classes of VP1 residues interact with +RNA to mediate template recognition and dsRNA synthesis yet function in concert to promote viral RNA replication at appropriate times and rates.     

Simplified RNA secondary structure mapping by automation of SHAPE data analysis

SHAPE (Selective 2'-hydroxyl acylation analysed by primer extension) technology has emerged as one of the leading methods of determining RNA secondary structure at the nucleotide level. A significant bottleneck in using SHAPE is the complex and time-consuming data processing that is required. We present here a modified data collection method and a series of algorithms, embodied in a program entitled Fast Analysis of SHAPE traces (FAST), which significantly reduces processing time. We have used this method to resolve the secondary structure of the first ~900 nt of the hepatitis C virus (HCV) genome, including the entire core gene. We have also demonstrated the ability of SHAPE/FAST to detect the binding of a small molecule inhibitor to the HCV internal ribosomal entry site (IRES). In conclusion, FAST allows for high-throughput data processing to match the current high-throughput generation of data possible with SHAPE, reducing the barrier to determining the structure of RNAs of interest.     

Selective 2'-hydroxyl acylation analyzed by primer extension (SHAPE): quantitative RNA structure analysis at single nucleotide resolution

Selective 2'-hydroxyl acylation analyzed by primer extension (SHAPE) interrogates local backbone flexibility in RNA at single-nucleotide resolution under diverse solution environments. Flexible RNA nucleotides preferentially sample local conformations that enhance the nucleophilic reactivity of 2'-hydroxyl groups toward electrophiles, such as N-methylisatoic anhydride (NMIA). Modified sites are detected as stops in an optimized primer extension reaction, followed by electrophoretic fragment separation. SHAPE chemistry scores local nucleotide flexibility at all four ribonucleotides in a single experiment and discriminates between base-paired versus unconstrained or flexible residues with a dynamic range of 20-fold or greater. Quantitative SHAPE reactivity information can be used to establish the secondary structure of an RNA, to improve the accuracy of structure prediction algorithms, to monitor structural differences between related RNAs or a single RNA in different states, and to detect ligand binding sites. SHAPE chemistry rarely needs significant optimization and requires two days to complete for an RNA of 100-200 nucleotides.     

The RNA chaperone StpA enables fast RNA refolding by destabilization of mutually exclusive base pairs within competing secondary structure elements

In bacteria RNA gene regulatory elements refold dependent on environmental clues between two or more long-lived conformational states each associated with a distinct regulatory state. The refolding kinetics are strongly temperature-dependent and especially at lower temperatures they reach timescales that are biologically not accessible. To overcome this problem, RNA chaperones have evolved. However, the precise molecular mechanism of how these proteins accelerate RNA refolding reactions remains enigmatic. Here we show how the RNA chaperone StpA of Escherichia coli leads to an acceleration of a bistable RNA’s refolding kinetics through the selective destabilization of key base pairing interactions. We find in laser assisted real-time NMR experiments on photocaged bistable RNAs that the RNA chaperone leads to a two-fold increase in refolding rates at low temperatures due to reduced stability of ground state conformations. Further, we can show that upon interaction with StpA, base pairing interactions in the bistable RNA are modulated to favor refolding through the dominant pseudoknotted transition pathway. Our results shed light on the molecular mechanism of the interaction between RNA chaperones and bistable RNAs and are the first step into a functional classification of chaperones dependent on their biophysical mode of operation.     

In-gel probing of individual RNA conformers within a mixed population reveals a dimerization structural switch in the HIV-1 leader

Definitive secondary structural mapping of RNAs in vitro can be complicated by the presence of more than one structural conformer or multimerization of some of the molecules. Until now, probing a single structure of conformationally flexible RNA molecules has typically relied on introducing stabilizing mutations or adjusting buffer conditions or RNA concentration. Here, we present an in-gel SHAPE (selective 2′OH acylation analysed by primer extension) approach, where a mixed structural population of RNA molecules is separated by non-denaturing gel electrophoresis and the conformers are individually probed within the gel matrix. Validation of the technique using a well-characterized RNA stem-loop structure, the HIV-1 trans-activation response element, showed that authentic structure was maintained and that the method was accurate and highly reproducible. To further demonstrate the utility of in-gel SHAPE, we separated and examined monomeric and dimeric species of the HIV-1 packaging signal RNA. Extensive differences in acylation sensitivity were seen between monomer and dimer. The results support a recently proposed structural switch model of RNA genomic dimerization and packaging, and demonstrate the discriminatory power of in-gel SHAPE.     

Four nucleotides are the minimal requirement for RNA recognition by rotavirus non-structural protein NSP3.

The interaction of the group A rotavirus non-structural protein NSP3 (NSP3A) with RNA has been studied in vitro. Using semi-purified NSP3A protein expressed by a recombinant baculovirus and in vitro synthesized RNA, we determined by UV cross-linking and gel retardation assays that NSP3A binds, in a sequence-specific manner, the consensus sequence (AUGUGACC) present on the 3' ends of all group A rotavirus mRNAs. Using short oligoribonucleotides, we established that the minimal RNA sequence required for binding of NSP3A is GACC. Modifications of the UGACC oligonucleotide sequence impaired binding of the protein to the RNA. Furthermore, the recombinant NSP3 protein from rotavirus group C showed specificity for the 3' end consensus sequence (AUGUGGCU) of only group C mRNAs. Sequence analysis of the NSP3 proteins did not reveal significant homologies with other RNA binding proteins, thus the NSP3 proteins of rotaviruses are the prototypes of a new kind of sequence-specific RNA binding protein.     

Influence of nucleotide identity on ribose 2′-hydroxyl reactivity in RNA

Hydroxyl-selective electrophiles, including N-methylisatoic anhydride (NMIA) and 1-methyl-7-nitroisatoic anhydride (1M7), are broadly useful for RNA structure analysis because they react preferentially with the ribose 2′-OH group at conformationally unconstrained or flexible nucleotides. Each nucleotide in an RNA has the potential to form an adduct with these reagents to yield a comprehensive, nucleotide-resolution, view of RNA structure. However, it is possible that factors other than local structure modulate reactivity. To evaluate the influence of base identity on the intrinsic reactivity of each nucleotide, we analyze NMIA and 1M7 reactivity using four distinct RNAs, under both native and denaturing conditions. We show that guanosine and adenosine residues have identical intrinsic 2′-hydroxyl reactivities at pH 8.0 and are 1.4 and 1.7 times more reactive than uridine and cytidine, respectively. These subtle, but statistically significant, differences do not impact the ability of selective 2′-hydroxyl acylation analyzed by primer extension-based (SHAPE) methods to establish an RNA secondary structure or monitor RNA folding in solution because base-specific influences are much smaller than the reactivity differences between paired and unpaired nucleotides.     

Selective 2'-hydroxyl acylation analyzed by protection from exoribonuclease (RNase-detected SHAPE) for direct analysis of covalent adducts and of nucleotide flexibility in RNA

RNA SHAPE chemistry yields quantitative, single-nucleotide resolution structural information based on the reaction of the 2'-hydroxyl group of conformationally flexible nucleotides with electrophilic SHAPE reagents. However, SHAPE technology has been limited by the requirement that sites of RNA modification be detected by primer extension. Primer extension results in loss of information at both the 5' and 3' ends of an RNA and requires multiple experimental steps. Here we describe RNase-detected SHAPE that uses a processive, 3'→5' exoribonuclease, RNase R, to detect covalent adducts in 5'-end-labeled RNA in a one-tube experiment. RNase R degrades RNA but stops quantitatively three and four nucleotides 3' of a nucleotide containing a covalent adduct at the ribose 2'-hydroxyl or the pairing face of a nucleobase, respectively. We illustrate this technology by characterizing ligand-induced folding for the aptamer domain of the Escherichia coli thiamine pyrophosphate riboswitch RNA. RNase-detected SHAPE is a facile, two-day approach that can be used to analyze diverse covalent adducts in any RNA molecule, including short RNAs not amenable to analysis by primer extension and RNAs with functionally important structures at their 5' or 3' ends.     

Structure-Based Alignment and Consensus Secondary Structures for Three HIV-Related RNA Genomes

HIV and related primate lentiviruses possess single-stranded RNA genomes. Multiple regions of these genomes participate in critical steps in the viral replication cycle, and the functions of many RNA elements are dependent on the formation of defined structures. The structures of these elements are still not fully understood, and additional functional elements likely exist that have not been identified. In this work, we compared three full-length HIV-related viral genomes: HIV-1_NL4-3, SIVcpz, and SIVmac (the latter two strains are progenitors for all HIV-1 and HIV-2 strains, respectively). Model-free RNA structure comparisons were performed using whole-genome structure information experimentally derived from nucleotide-resolution SHAPE reactivities. Consensus secondary structures were constructed for strongly correlated regions by taking into account both SHAPE probing structural data and nucleotide covariation information from structure-based alignments. In these consensus models, all known functional RNA elements were recapitulated with high accuracy. In addition, we identified multiple previously unannotated structural elements in the HIV-1 genome likely to function in translation, splicing and other replication cycle processes; these are compelling targets for future functional analyses. The structure-informed alignment strategy developed here will be broadly useful for efficient RNA motif discovery.