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Many lncRNAs have been discovered using transcriptomic data; however, it is unclear what fraction of lncRNAs is functional and what structural properties affect their phenotype. MUNC lncRNA (also known as

Chemical probing experiments, coupled with empirically determined free energy change relationships, can enable accurate modeling of the secondary structures of diverse and complex RNAs. A current frontier lies in modeling large and structurally heterogeneous transcripts, including complex eukaryotic RNAs. To validate and improve on experimentally driven approaches for modeling large transcripts, we obtained high-quality SHAPE data for the protein-free human 18S and 28S ribosomal RNAs (rRNAs). To our surprise, SHAPE-directed structure models for the human rRNAs poorly matched accepted structures. Analysis of predicted rRNA structures based on low-SHAPE and low-entropy (lowSS) metrics revealed that, whereas ∼75% of Escherichia coli rRNA sequences form well-determined lowSS secondary structure, only ∼40% of the human rRNAs do. Critically, regions of the human rRNAs that specifically fold into well-determined lowSS structures were modeled to high accuracy using SHAPE data. This work reveals that eukaryotic rRNAs are more unfolded than are those of prokaryotic rRNAs and indeed are largely unfolded overall, likely reflecting increased protein dependence for eukaryotic ribosome structure. In addition, those regions and substructures that are well-determined can be identified de novo and successfully modeled by SHAPE-directed folding.

The functions of most long non-coding RNAs (lncRNAs) are unknown. In contrast to proteins, lncRNAs with similar functions often lack linear sequence homology; thus, the identification of function in one lncRNA rarely informs the identification of function in others. We developed a sequence comparison method to deconstruct linear sequence relationships in lncRNAs and evaluate similarity based on the abundance of short motifs called k-mers. We found that lncRNAs of related function often had similar k-mer profiles despite lacking linear homology, and that k-mer profiles correlated with protein binding to lncRNAs and with their subcellular localization. Using a novel assay to quantify Xist-like regulatory potential, we directly demonstrated that evolutionarily unrelated lncRNAs can encode similar function through different spatial arrangements of related sequence motifs. K-mer-based classification is a powerful approach to detect recurrent relationships between sequence and function in lncRNAs.

The HIV-1 RNA genome contains complex structures with many structural elements playing regulatory roles during viral replication. A recent study has identified multiple RNA structures with unknown functions that are conserved among HIV-1 and two simian immunodeficiency viruses. To explore the roles of these conserved RNA structures, we introduced synonymous mutations into the HIV-1 genome to disrupt each structure. These mutants exhibited similar particle production, viral infectivity, and replication kinetics relative to the parent NL4-3 virus. However, when replicating in direct competition with the wild-type NL4-3 virus, mutations of RNA structures at inter-protein domain junctions can cause fitness defects. These findings reveal the ability of HIV-1 to tolerate changes in its sequences, even in apparently highly conserved structures, which permits high genetic diversity in HIV-1 population. Our results also suggest that some conserved RNA structures may function to fine-tune viral replication.

Summary: Long non-coding RNAs (lncRNAs) are critical regulators of numerous physiological processes and diseases, especially cancers. However, development of lncRNA-based therapies is limited because the mechanisms of many lncRNAs are obscure, and interactions with functional partners, including proteins, remain uncharacterized. The lncRNA SLNCR1 binds to and regulates the androgen receptor (AR) to mediate melanoma invasion and proliferation in an androgen-independent manner. Here, we use biochemical analyses coupled with selective 2′-hydroxyl acylation analyzed by primer extension (SHAPE) RNA structure probing to show that the N-terminal domain of AR binds a pyrimidine-rich motif in an unstructured region of SLNCR1. This motif is predictive of AR binding, as we identify an AR-binding motif in lncRNA HOXA11-AS-203. Oligonucleotides that bind either the AR N-terminal domain or the AR RNA motif block the SLNCR1-AR interaction and reduce SLNCR1-mediated melanoma invasion. Delivery of oligos that block SLNCR1-AR interaction thus represent a plausible therapeutic strategy. : Androgen receptor (AR)-RNA complexes have been implicated in cancer, including melanoma. Schmidt et al. demonstrate that AR binds a single strand sequence in the long non-coding RNA (lncRNA) SLNCR. Point mutations or oligonucleotides that abrogate AR binding to SLNCR block melanoma invasion, suggesting that targeting lncRNA-protein complexes holds therapeutic promise. Keywords: long non-coding RNA, hormone receptor, androgen receptor, HOXA11AS, LINC00673, antisense oligos, melanoma, invasion, RNA scaffold, SHAPE

RNA and membraneless organelles Membraneless compartments can form in cells through liquidliquid phase separation (see the Perspective by Polymenidou). But what prevents these cellular condensates from randomly fusing together? Using the RNA-binding protein (RBP) Whi3, Langdon et al. demonstrated that the secondary structure of different RNA components determines the distinct biophysical and biological properties of the two types of condensates that Whi3 forms. Several RBPs, such as FUS and TDP43, contain prion-like domains and are linked to neurodegenerative diseases. These RBPs are usually soluble in the nucleus but can form pathological aggregates in the cytoplasm. Maharana et al. showed that local RNA concentrations determine distinct phase separation behaviors in different subcellular locations. The higher RNA concentrations in the nucleus act as a buffer to prevent phase separation of RBPs; when mislocalized to the cytoplasm, lower RNA concentrations trigger aggregation. Science , this issue p. 922 , p. 918 ; see also p. 859

There are large differences between the cellular environment and the conditions widely used to study RNA in vitro. SHAPE RNA structure probing in Escherichia coli cells has shown that the cellular environment stabilizes both long-range and local tertiary interactions in the adenine riboswitch aptamer domain. Synthetic crowding agents are widely used to understand the forces that stabilize RNA structure and in efforts to recapitulate the cellular environment under simplified experimental conditions. Here, we studied the structure and ligand binding ability of the adenine riboswitch in the presence of the macromolecular crowding agent, polyethylene glycol (PEG). Ethylene glycol and low-molecular mass PEGs destabilized RNA structure and caused the riboswitch to sample secondary structures different from those observed in simple buffered solutions or in cells. In the presence of larger PEGs, longer-range loop-loop interactions were more similar to those in cells than in buffer alone, consistent with prior work showing that larger PEGs stabilize compact RNA states. Ligand affinity was weakened by low-molecular mass PEGs but increased with high-molecular mass PEGs, indicating that PEG cosolvents exert complex chemical and steric effects on RNA structure. Regardless of polymer size, however, nucleotide-resolution structural characteristics observed in cells were not recapitulated in PEG solutions. Our results reveal that the cellular environment is difficult to recapitulate in vitro; mimicking the cellular state will likely require a combination of crowding agents and other chemical species.

Hepatitis C virus (HCV) infects over 170 million people worldwide and is a leading cause of liver disease and cancer. The virus has a 9,650-nt, single-stranded, messenger-sense RNA genome that is infectious as an independent entity. The RNA genome has evolved in response to complex selection pressures, including the need to maintain structures that facilitate replication and to avoid clearance by cell-intrinsic immune processes. Here we used high-throughput, single-nucleotide resolution information to generate and functionally test data-driven structural models for three diverse HCV RNA genomes. We identified, de novo, multiple regions of conserved RNA structure, including all previously characterized cis-acting regulatory elements and also multiple novel structures required for optimal viral fitness. Well-defined RNA structures in the central regions of HCV genomes appear to facilitate persistent infection by masking the genome from RNase L and double-stranded RNA-induced innate immune sensors. This work shows how structure-first comparative analysis of entire genomes of a pathogenic RNA virus enables comprehensive and concise identification of regulatory elements and emphasizes the extensive interrelationships among RNA genome structure, viral biology, and innate immune responses.

Significance Protein and mRNA expression are in most cases poorly correlated, which suggests that the posttranscriptional regulatory program of a cell is an important component of gene expression. This regulatory network is still poorly understood, including how RNA structure quantitatively contributes to translational control. We present here a series of structural and functional experiments that together allow us to derive a quantitative, structure-dependent model of translation that accurately predicts translation efficiency in reporter assays and primary human tissue for a complex and medically important protein, α-1-antitrypsin. Our model demonstrates the importance of accurate, experimentally derived RNA structural models partnered with Kozak sequence information to explain protein expression and suggests a strategy by which α-1-antitrypsin expression may be increased in diseased individuals., Chronic obstructive pulmonary disease (COPD) affects over 65 million individuals worldwide, where α-1-antitrypsin deficiency is a major genetic cause of the disease. The α-1-antitrypsin gene, SERPINA1, expresses an exceptional number of mRNA isoforms generated entirely by alternative splicing in the 5′-untranslated region (5′-UTR). Although all SERPINA1 mRNAs encode exactly the same protein, expression levels of the individual mRNAs vary substantially in different human tissues. We hypothesize that these transcripts behave unequally due to a posttranscriptional regulatory program governed by their distinct 5′-UTRs and that this regulation ultimately determines α-1-antitrypsin expression. Using whole-transcript selective 2′-hydroxyl acylation by primer extension (SHAPE) chemical probing, we show that splicing yields distinct local 5′-UTR secondary structures in SERPINA1 transcripts. Splicing in the 5′-UTR also changes the inclusion of long upstream ORFs (uORFs). We demonstrate that disrupting the uORFs results in markedly increased translation efficiencies in luciferase reporter assays. These uORF-dependent changes suggest that α-1-antitrypsin protein expression levels are controlled at the posttranscriptional level. A leaky-scanning model of translation based on Kozak translation initiation sequences alone does not adequately explain our quantitative expression data. However, when we incorporate the experimentally derived RNA structure data, the model accurately predicts translation efficiencies in reporter assays and improves α-1-antitrypsin expression prediction in primary human tissues. Our results reveal that RNA structure governs a complex posttranscriptional regulatory program of α-1-antitrypsin expression. Crucially, these findings describe a mechanism by which genetic alterations in noncoding gene regions may result in α-1-antitrypsin deficiency.

Many biological processes are RNA-mediated, but higher-order structures for most RNAs are unknown, which makes it difficult to understand how RNA structure governs function. Here we describe selective 2'-hydroxyl acylation analyzed by primer extension and mutational profiling (SHAPE-MaP) that makes possible de novo and large-scale identification of RNA functional motifs. Sites of 2'-hydroxyl acylation by SHAPE are encoded as noncomplementary nucleotides during cDNA synthesis, as measured by massively parallel sequencing. SHAPE-MaP-guided modeling identified greater than 90% of accepted base pairs in complex RNAs of known structure, and we used it to define a new model for the HIV-1 RNA genome. The HIV-1 model contains all known structured motifs and previously unknown elements, including experimentally validated pseudoknots. SHAPE-MaP yields accurate and high-resolution secondary-structure models, enables analysis of low-abundance RNAs, disentangles sequence polymorphisms in single experiments and will ultimately democratize RNA-structure analysis.

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.

There are large differences between the intracellular environment and the conditions widely used to study RNA structure and function in vitro. To assess the effects of the crowded cellular environment on RNA, we examined the structure and ligand binding function of the adenine riboswitch aptamer domain in healthy, growing Escherichia coli cells at single-nucleotide resolution on the minute time scale using SHAPE (selective 2'-hydroxyl acylation analyzed by primer extension). The ligand-bound aptamer structure is essentially the same in cells and in buffer at 1 mM Mg(2+), the approximate Mg(2+) concentration we measured in cells. In contrast, the in-cell conformation of the ligand-free aptamer is much more similar to the fully folded ligand-bound state. Even adding high Mg(2+) concentrations to the buffer used for in vitro analyses did not yield the conformation observed for the free aptamer in cells. The cellular environment thus stabilizes the aptamer significantly more than does Mg(2+) alone. Our results show that the intracellular environment has a large effect on RNA structure that ultimately favors highly organized conformations.

RNA chaperones are ubiquitous, heterogeneous proteins essential for RNA structural biogenesis and function. We investigated the mechanism of chaperone-mediated RNA folding by following the time-resolved dimerization of the packaging domain of a retroviral RNA at nucleotide resolution. In the absence of the nucleocapsid (NC) chaperone, dimerization proceeded through multiple, slow-folding intermediates. In the presence of NC, dimerization occurred rapidly through a single structural intermediate. The RNA binding domain of heterogeneous nuclear ribonucleoprotein A1 protein, a structurally unrelated chaperone, also accelerated dimerization. Both chaperones interacted primarily with guanosine residues. Replacing guanosine with more weakly pairing inosine yielded an RNA that folded rapidly without a facilitating chaperone. These results show that RNA chaperones can simplify RNA folding landscapes by weakening intramolecular interactions involving guanosine and explain many RNA chaperone activities.

We report the results of a first, collective, blind experiment in RNA three-dimensional (3D) structure prediction, encompassing three prediction puzzles. The goals are to assess the leading edge of RNA structure prediction techniques; compare existing methods and tools; and evaluate their relative strengths, weaknesses, and limitations in terms of sequence length and structural complexity. The results should give potential users insight into the suitability of available methods for different applications and facilitate efforts in the RNA structure prediction community in ongoing efforts to improve prediction tools. We also report the creation of an automated evaluation pipeline to facilitate the analysis of future RNA structure prediction exercises.

All retroviral genomic RNAs contain a cis-acting packaging signal by which dimeric genomes are selectively packaged into nascent virions. However, it is not understood how Gag (the viral structural protein) interacts with these signals to package the genome with high selectivity. We probed the structure of murine leukemia virus RNA inside virus particles using SHAPE, a high-throughput RNA structure analysis technology. These experiments showed that NC (the nucleic acid binding domain derived from Gag) binds within the virus to the sequence UCUG-UR-UCUG. Recombinant Gag and NC proteins bound to this same RNA sequence in dimeric RNA in vitro; in all cases, interactions were strongest with the first U and final G in each UCUG element. The RNA structural context is critical: High-affinity binding requires base-paired regions flanking this motif, and two UCUG-UR-UCUG motifs are specifically exposed in the viral RNA dimer. Mutating the guanosine residues in these two motifs—only four nucleotides per genomic RNA—reduced packaging 100-fold, comparable to the level of nonspecific packaging. These results thus explain the selective packaging of dimeric RNA. This paradigm has implications for RNA recognition in general, illustrating how local context and RNA structure can create information-rich recognition signals from simple single-stranded sequence elements in large RNAs.

Selective 2'-hydroxyl acylation analyzed by primer extension (SHAPE) is a powerful approach for characterizing RNA structure and dynamics at single-nucleotide resolution. However, SHAPE technology is limited, sometimes severely, because primer extension detection obscures structural information for approximately 15 nts at the 5' end and 40-60 nts at the 3' end of the RNA. Moreover, detection by primer extension is more complex than the actual structure-selective chemical interrogation step. Here we quantify covalent adducts in RNA directly by adduct-inhibited exoribonuclease degradation. RNA 2'-O-adducts block processivity of a 3'-->5' exoribonuclease, RNase R, to produce fragments that terminate three nucleotides 3' of the modification site. We analyzed the structure of the native thiamine pyrophosphate (TPP) riboswitch aptamer domain and identified large changes in local nucleotide dynamics and global RNA structure upon ligand binding. In addition to numerous changes that can be attributed to ligand recognition, we identify a single nucleotide bulge register shift, distant from the binding site, that stabilizes the ligand-bound structure. Selective 2'-hydroxyl acylation analyzed by protection from exoribonuclease (RNase-detected SHAPE) should prove broadly useful for facile structural analysis of small noncoding RNAs and for RNAs that have functionally critical structures at their 5' and 3' ends.

Retroviral genomes are dimeric, comprised of two sense-strand RNAs linked at their 5′ ends by noncovalent base pairing and tertiary interactions. Viral maturation involves large-scale morphological changes in viral proteins and in genomic RNA dimer structures to yield infectious virions. Structural studies have largely focused on simplified in vitro models of genomic RNA dimers even though the relationship between these models and authentic viral RNA is unknown. We evaluate the secondary structure of the minimal dimerization domain in genomes isolated from Moloney murine leukemia virions using a quantitative and single nucleotide resolution RNA structure analysis technology (selective 2′-hydroxyl acylation analyzed by primer extension, or SHAPE). Results are consistent with an architecture in which the RNA dimer is stabilized by four primary interactions involving two sets of intermolecular base pairs and two loop-loop interactions. The dimerization domain can independently direct its own folding since heating and refolding reproduce the same structure as visualized in genomic RNA isolated from virions. Authentic ex virio RNA has a SHAPE reactivity profile similar to that of a simplified transcript dimer generated in vitro , with the important exception of a region that appears to form a compact stem-loop only in the virion-isolated RNA. Finally, we analyze the conformational changes that accompany folding of monomers into dimers in vitro . These experiments support well-defined structural models for an authentic dimerization domain and also emphasize that many features of mature genomic RNA dimers can be reproduced in vitro using properly designed, simplified RNAs.

RNA selective 2'-hydroxyl acylation analyzed by primer extension (SHAPE) chemistry exploits the discovery that conformationally dynamic nucleotides preferentially adopt configurations that facilitate reaction between the 2'-OH group and a hydroxyl-selective electrophile, such as benzoyl cyanide (BzCN), to form a 2'-O-adduct. BzCN is ideally suited for quantitative, time-resolved analysis of RNA folding and ribonucleoprotein (RNP) assembly mechanisms because this reagent both reacts with flexible RNA nucleotides and also undergoes auto-inactivating hydrolysis with a half-life of 0.25 s at 37 degrees C. RNA folding is initiated by addition of Mg(2+) or protein, or other change in solution conditions, and nucleotide resolution structural images are obtained by adding aliquots of the evolving reaction to BzCN and then 'waiting' for 1 second. Sites of the 2'-O-adduct formation are subsequently scored as stops to primer extension using reverse transcriptase. This time-resolved SHAPE protocol makes it possible to obtain 1-second structural snapshots in time-resolved kinetic studies for RNAs of arbitrary length and complexity in a straightforward and concise experiment.

Analysis of the long-range architecture of RNA is a challenging experimental and computational problem. Local nucleotide flexibility, which directly reports underlying base pairing and tertiary interactions in an RNA, can be comprehensively assessed at single nucleotide resolution using high-throughput selective 2′-hydroxyl acylation analyzed by primer extension (hSHAPE). hSHAPE resolves structure-sensitive chemical modification information by high-resolution capillary electrophoresis and typically yields quantitative nucleotide flexibility information for 300–650 nucleotides (nt) per experiment. The electropherograms generated in hSHAPE experiments provide a wealth of structural information; however, significant algorithmic analysis steps are required to generate quantitative and interpretable data. We have developed a set of software tools called ShapeFinder to make possible rapid analysis of raw sequencer data from hSHAPE, and most other classes of nucleic acid reactivity experiments. The algorithms in ShapeFinder (1) convert measured fluorescence intensity to quantitative cDNA fragment amounts, (2) correct for signal decay over read lengths extending to 600 nts or more, (3) align reactivity data to the known RNA sequence, and (4) quantify per nucleotide reactivities using whole-channel Gaussian integration. The algorithms and user interface tools implemented in ShapeFinder create new opportunities for tackling ambitious problems involving high-throughput analysis of structure–function relationships in large RNAs.

The mammalian mitochondrial genome encodes 13 proteins, which are synthesized at the direction of nine monocistronic and two dicistronic mRNAs. These mRNAs lack both 5′ and 3′ untranslated regions. The mechanism by which the specialized mitochondrial translational apparatus locates start codons and initiates translation of these leaderless mRNAs is currently unknown. To better understand this mechanism, the secondary structures near the start codons of all 13 open reading frames have been analyzed using RNA SHAPE chemistry. The extent of structure in these mRNAs as assessed experimentally is distinctly lower than would be predicted by current algorithms based on free energy minimization alone. We find that the 5′ ends of all mitochondrial mRNAs are highly unstructured. The first 35 nucleotides for all mitochondrial mRNAs form structures with free energies less favorable than −3 kcal/mol, equal to or less than a single typical base pair. The start codons, which lie at the very 5′ ends of these mRNAs, are accessible within single stranded motifs in all cases, making them potentially poised for ribosome binding. These data are consistent with a model in which the specialized mitochondrial ribosome preferentially allows passage of unstructured 5′ sequences into the mRNA entrance site to participate in translation initiation.

The emergence of catalysis in early genetic polymers such as RNA is considered a key transition in the origin of life, pre-dating the appearance of protein enzymes. DNA also demonstrates the capacity to fold into three-dimensional structures and form catalysts in vitro. However, to what degree these natural biopolymers comprise functionally privileged chemical scaffolds for folding or the evolution of catalysis is not known. The ability of synthetic genetic polymers (XNAs) with alternative backbone chemistries not found in nature to fold into defined structures and bind ligands raises the possibility that these too might be capable of forming catalysts (XNAzymes). Here we report the discovery of such XNAzymes, elaborated in four different chemistries (arabino nucleic acids, ANA; 2'-fluoroarabino nucleic acids, FANA; hexitol nucleic acids, HNA; and cyclohexene nucleic acids, CeNA) directly from random XNA oligomer pools, exhibiting in trans RNA endonuclease and ligase activities. We also describe an XNA-XNA ligase metalloenzyme in the FANA framework, establishing catalysis in an entirely synthetic system and enabling the synthesis of FANA oligomers and an active RNA endonuclease FANAzyme from its constituent parts. These results extend catalysis beyond biopolymers and establish technologies for the discovery of catalysts in a wide range of polymer scaffolds not found in nature. Evolution of catalysis independent of any natural polymer has implications for the definition of chemical boundary conditions for the emergence of life on Earth and elsewhere in the Universe. ispartof: Nature vol:518 issue:7539 pages:427-430 ispartof: location:England status: published

Discovery and characterization of functional RNA structures remains challenging due to deficiencies in de novo secondary structure modeling. Here we describe a dynamic programming approach for model-free sequence comparison that incorporates high-throughput chemical probing data. Based on SHAPE probing data alone, ribosomal RNAs (rRNAs) from three diverse organisms – the eubacteria E. coli and C. difficile and the archeon H. volcanii – could be aligned with accuracies comparable to alignments based on actual sequence identity. When both base sequence identity and chemical probing reactivities were considered together, accuracies improved further. Derived sequence alignments and chemical probing data from protein-free RNAs were then used as pseudo-free energy constraints to model consensus secondary structures for the 16S and 23S rRNAs. There are critical differences between these experimentally-informed models and currently accepted models, including in the functionally important neck and decoding regions of the 16S rRNA. We infer that the 16S rRNA has evolved to undergo large-scale changes in base pairing as part of ribosome function. As high-quality RNA probing data become widely available, structurally-informed sequence alignment will become broadly useful for de novo motif and function discovery., Author Summary Despite the clear functional importance of structure in RNA molecules, it remains very difficult to correctly identify and annotate similar RNA structures because their sequences are often poorly conserved even for RNAs that form very similar higher-order structures. A solution is to use a metric that identifies structural motifs but this, too, is difficult because RNA structure modeling based on sequence alone is generally not very accurate. In this work, we use SHAPE chemical probing to obtain model-free information about RNA structure and then exploit this information to align RNAs by sequence. We show for ribosomal RNAs that sequence alignments based on SHAPE experimental information alone are as accurate as those that actually use sequence information. In addition, we identify regions in the 16S ribosomal RNA that form conserved secondary structures that are different from currently accepted models. These differences, rather than being errors, reveal sites of conformational flexibility that may underlie mechanistic functions in ribosome assembly and translation regulation. We anticipate that structure-informed sequence alignment and structure modeling will become broadly useful tools in RNA function analysis.

Retroviral genomes are assembled from two sense-strand RNAs by noncovalent interactions at their 5' ends, forming a dimer. The RNA dimerization domain is a potential target for antiretroviral therapy and represents a compelling RNA folding problem. The fundamental dimerization unit for the Moloney murine sarcoma gamma retrovirus spans a 170-nucleotide minimal dimerization active sequence. In the dimer, two self-complementary sequences, PAL1 and PAL2, form intermolecular duplexes, and an SL1-SL2 (stem-loop) domain forms loop-loop base pairs, mediated by GACG tetraloops, and extensive tertiary interactions. To develop a framework for assembly of the retroviral RNA dimer, we quantified the stability of and established nucleotide resolution secondary structure models for sequence variants in which each motif was compromised. Base pairing and tertiary interactions between SL1-SL2 domains contribute a large free energy increment of -10 kcal/mol. In contrast, even though the PAL1 and PAL2 intermolecular duplexes span 10 and 16 bp in the dimer, respectively, they contribute only -2.5 kcal/mol to stability, roughly equal to a single new base pair. First, these results emphasize that the energetic costs for disrupting interactions in the monomer state nearly balance the PAL1 and PAL2 base pairing interactions that form in the dimer. Second, intermolecular duplex formation plays a biological role distinct from simply stabilizing the structure of the retroviral genomic RNA dimer.

Retroviral genomes contain two sense-strand RNAs that are noncovalently linked at their 5' ends, forming a dimer. Establishing a structure for this dimer is an obligatory first step toward understanding the fundamental role of the dimeric RNA in retroviral biology. We developed a secondary structure model for the minimal dimerization active sequence (MiDAS) for the Moloney murine sarcoma virus in the final dimer state using selective 2'-hydroxyl acylation analyzed by primer extension (SHAPE). In this model, two self-complementary, or palindromic, sequences (PAL1 and PAL2) form extended intermolecular duplexes of 10 and 16 base pairs, respectively. The monomeric starting state was shown previously to contain a flexible domain in which nucleotides do not form stable interactions with other parts of the RNA. In the final dimer state, portions of this initial flexible domain form stable base pairs, while previously base-paired elements lie in a new flexible domain. Thus, partially overlapping and structurally well-defined flexible domains are prominent features of both monomer and dimer states. We then used hydroxyl radical cleavage experiments to characterize the global architecture of the dimer state. Extensive regions, including portions of both PAL1 and PAL2, are occluded from solvent-based cleavage indicating that the MiDAS domain does not function simply as a collection of autonomous secondary structure elements. Instead, the retroviral dimerization domain adopts a compact architecture characterized by close packing of its constituent helices.

LAGLIDADG endonucleases bind across adjacent major grooves via a saddle-shaped surface and catalyze DNA cleavage. Some LAGLIDADG proteins, called maturases, facilitate splicing by group I introns, raising the issue of how a DNA-binding protein and an RNA have evolved to function together. In this report, crystallographic analysis shows that the global architecture of the bI3 maturase is unchanged from its DNA-binding homologs; in contrast, the endonuclease active site, dispensable for splicing facilitation, is efficiently compromised by a lysine residue replacing essential catalytic groups. Biochemical experiments show that the maturase binds a peripheral RNA domain 50 A from the splicing active site, exemplifying long-distance structural communication in a ribonucleoprotein complex. The bI3 maturase nucleic acid recognition saddle interacts at the RNA minor groove; thus, evolution from DNA to RNA function has been mediated by a switch from major to minor groove interaction.

Current models assume that RNA folding is strongly hierarchical such that the base-paired secondary structure is more stable than and forms independently of the tertiary structure. This model has been difficult to test due to the experimental inability to interrogate the local environment at every nucleotide as a comprehensive function of the RNA folding state. Reaction of an RNA 2'-hydroxyl group with N-methylisatoic anhydride to form a nucleotide 2'-ester is governed by the extent to which the nucleotide forms base pairing or tertiary interactions. Selective 2'-Hydroxyl Acylation analyzed by Primer Extension (SHAPE) is shown to be an RNA analogue of the protein hydrogen exchange experiment. Single nucleotide resolution SHAPE analysis emphasizes a complexity for the unfolding of tRNA(Asp) transcripts that is not anticipated by current models for RNA folding. We quantify six well-defined transitions for tRNA(Asp) transcripts between 35 and75 degrees C, including asymmetric unfolding of the two strands in a single helix, multistep loss of tertiary interactions, and a multihelix conformational shift. The three lowest temperature transitions each involve coupled interactions between the secondary and tertiary structure. Thus, even for this simple RNA, multiple nonhierarchical and complex interactions dominate the equilibrium transitions most accessible from the native state.

We describe structural analysis of RNAs by SHAPE chemical probing. RNAs are treated with 1-methyl-7-nitroisatoic anhydride (1M7), a reagent that detects local nucleotides flexibility, and N-methylisatoic anhydride (NMIA) and 1-methyl-6-nitroisatoic anhydride (1M6), reagents which together detect higher-order and non-canonical interactions. Chemical adducts are detected as stops during reverse transcriptase-mediated primer extension. Probing information can be used to infer conformational changes and ligand binding, and to develop highly accurate models of RNA secondary structures.

Native folding and splicing by the Saccharomyces cerevisiae mitochondrial bI5 group I intron RNA is facilitated by both the S. cerevisiae CBP2 and Neurospora crassa CYT-18 protein cofactors. Both protein-bI5 RNA complexes splice at similar rates, suggesting that the RNA active site structure is similar in both ribonucleoproteins. In contrast, the two proteins assemble with the bI5 RNA by distinct mechanisms and bind opposing, but partially overlapping, sides of the group I intron catalytic core. Assembly with CBP2 is limited by a slow, unimolecular RNA folding step characterized by a negligible activation enthalpy. We show that assembly with CYT-18 shows four distinctive features. (1) CYT-18 binds stably to the bI5 RNA at the diffusion controlled limit, but assembly to a catalytically active RNA structure is still limited by RNA folding, as visualized directly using time-resolved footprinting. (2) This mechanism of rapid stable protein binding followed by subsequent assembly steps has a distinctive kinetic signature: the apparent ratio of koff to kon, determined in a partitioning experiment, differs from the equilibrium Kd by a large factor. (3) Assembly with CYT-18 is characterized by a large activation enthalpy, consistent with a rate limiting conformational rearrangement. (4) Because assembly from the kinetically trapped state is faster at elevated temperature, we can identify conditions where CYT-18 accelerates (catalyzes) bI5 RNA folding relative to assembly with CBP2. # 2001 Academic Press

Accurate RNA structure modeling is an important, incompletely solved, challenge. Single-nucleotide resolution SHAPE (selective 2'-hydroxyl acylation analyzed by primer extension) yields an experimental measurement of local nucleotide flexibility that can be incorporated as pseudo-free energy change constraints to direct secondary structure predictions. Prior work from our laboratory has emphasized both the overall accuracy of this approach and the need for nuanced interpretation of some apparent discrepancies between modeled and accepted structures. Recent studies by Das and colleagues [Kladwang et al., Biochemistry 50:8049 (2011) and Nat. Chem. 3:954 (2011)], focused on analyzing six small RNAs, yielded poorer RNA secondary structure predictions than expected based on prior benchmarking efforts. To understand the features that led to these divergent results, we re-examined four RNAs yielding the poorest results in this recent work – tRNAPhe, the adenine and cyclic-di-GMP riboswitches, and 5S rRNA. Most of the errors reported by Das and colleagues reflected non-standard experiment and data processing choices, and selective scoring rules. For two RNAs, tRNAPhe and the adenine riboswitch, secondary structure predictions are nearly perfect if no experimental information is included but were rendered inaccurate by the Das and colleagues SHAPE data. When best practices were used, single-sequence SHAPE-directed secondary structure modeling recovered ~93% of individual base pairs and greater than 90% of helices in the four RNAs, essentially indistinguishable from the mutate-and-map approach with the exception of a single helix in the 5S rRNA. The field of experimentally-directed RNA secondary structure prediction is entering a phase focused on the most difficult prediction challenges. We outline five constructive principles for guiding this field forward.

RNA secondary structure plays a central role in the replication and metabolism of all RNA viruses, including retroviruses like HIV-1. However, structures with known function represent only a fraction of the secondary structure reported for HIV-1NL4-3. One tool to assess the importance of RNA structures is to examine their conservation over evolutionary time. To this end, we used SHAPE to model the secondary structure of a second primate lentiviral genome, SIVmac239, which shares only 50% sequence identity at the nucleotide level with HIV-1NL4-3. Only about half of the paired nucleotides are paired in both genomic RNAs and, across the genome, just 71 base pairs form with the same pairing partner in both genomes. On average the RNA secondary structure is thus evolving at a much faster rate than the sequence. Structure at the Gag-Pro-Pol frameshift site is maintained but in a significantly altered form, while the impact of selection for maintaining a protein binding interaction can be seen in the conservation of pairing partners in the small RRE stems where Rev binds. Structures that are conserved between SIVmac239 and HIV-1NL4-3 also occur at the 5′ polyadenylation sequence, in the plus strand primer sites, PPT and cPPT, and in the stem-loop structure that includes the first splice acceptor site. The two genomes are adenosine-rich and cytidine-poor. The structured regions are enriched in guanosines, while unpaired regions are enriched in adenosines, and functionaly important structures have stronger base pairing than nonconserved structures. We conclude that much of the secondary structure is the result of fortuitous pairing in a metastable state that reforms during sequence evolution. However, secondary structure elements with important function are stabilized by higher guanosine content that allows regions of structure to persist as sequence evolution proceeds, and, within the confines of selective pressure, allows structures to evolve., Author Summary We have taken advantage of the rapid evolution of primate lentiviruses to assess the conservation of secondary structure in the viral RNA genome. We determined the structure of the SIVmac239 RNA genome to allow a detailed comparison with the previously determined structure of the HIV-1NL4-3 genome. In comparing the two structures, we find very few conserved base pairs with the same pairing partners, indicating that RNA structure is evolving even faster than the sequence. This suggests that most of the genome is in a metastable state that refolds during sequence evolution. Specific areas of structure that are required for function are maintained by the clustering of guanosines in the otherwise adenosine-rich genome, although the precise organization of the structure evolves. The strong effect of selection on maintainence of protein recognition sites can be seen in the conservation of pairing partners within the Rev binding sites in the RRE RNA. We propose that the more stable elements of RNA structure that are needed for function are susceptible to mutation during viral DNA synthesis. This causes the structures to evolve rapidly, yet still within the constricts of selective pressure, allowing maintenance of function.

Molecular modeling guided by experimentally derived structural information is an attractive approach for three-dimensional structure determination of complex RNAs that are not amenable to study by high-resolution methods. Hydroxyl radical probing (HRP), which is performed routinely in many laboratories, provides a measure of solvent accessibility at individual nucleotides. HRP measurements have, to date, only been used to evaluate RNA models qualitatively. Here we report the development of a quantitative structure refinement approach using HRP measurements to drive discrete molecular dynamics simulations for RNAs ranging in size from 80 to 230 nucleotides. We first used HRP reactivities to identify RNAs that form extensive helical packing interactions. For these RNAs, we achieved highly significant structure predictions given the inputs of RNA sequence and base pairing. This HRP-directed tertiary structure refinement approach generates robust structural hypotheses that are useful for guiding explorations of structure-function inter-relationships in RNA.

Higher-order structure influences critical functions in nearly all noncoding and coding RNAs. Most single-nucleotide resolution RNA structure determination technologies cannot be used to analyze RNA from scarce biological samples, like viral genomes. To make quantitative RNA structure analysis applicable to a much wider array of RNA structure-function problems, we developed and applied high-sensitivity selective 2'-hydroxyl acylation analyzed by primer extension (SHAPE) to structural analysis of authentic genomic RNA of the xenotropic murine leukemia virus-related virus (XMRV). For analysis of fluorescently labeled cDNAs generated in high-sensitivity SHAPE experiments, we developed a two-color capillary electrophoresis approach with zeptomole molecular detection limits and subfemtomole sensitivity for complete SHAPE experiments involving hundreds of individual RNA structure measurements. High-sensitivity SHAPE data correlated closely (R = 0.89) with data obtained by conventional capillary electrophoresis. Using high-sensitivity SHAPE, we determined the dimeric structure of the XMRV packaging domain, examined dynamic interactions between the packaging domain RNA and viral nucleocapsid protein inside virion particles, and identified the packaging signal for this virus. Despite extensive sequence differences between XMRV and the intensively studied Moloney murine leukemia virus, architectures of the regulatory domains are similar and reveal common principles of gammaretrovirus RNA genome packaging.

New regulatory roles continue to emerge for both natural and engineered noncoding RNAs, many of which have specific secondary and tertiary structures essential to their function. Thus there is a growing need to develop technologies that enable rapid characterization of structural features within complex RNA populations. We have developed a high-throughput technique, SHAPE-Seq, that can simultaneously measure quantitative, single nucleotide-resolution secondary and tertiary structural information for hundreds of RNA molecules of arbitrary sequence. SHAPE-Seq combines selective 2'-hydroxyl acylation analyzed by primer extension (SHAPE) chemistry with multiplexed paired-end deep sequencing of primer extension products. This generates millions of sequencing reads, which are then analyzed using a fully automated data analysis pipeline, based on a rigorous maximum likelihood model of the SHAPE-Seq experiment. We demonstrate the ability of SHAPE-Seq to accurately infer secondary and tertiary structural information, detect subtle conformational changes due to single nucleotide point mutations, and simultaneously measure the structures of a complex pool of different RNA molecules. SHAPE-Seq thus represents a powerful step toward making the study of RNA secondary and tertiary structures high throughput and accessible to a wide array of scientific pursuits, from fundamental biological investigations to engineering RNA for synthetic biological systems.

Nucleic acids are particularly amenable to structural characterization using chemical and enzymatic probes. Each individual structure mapping experiment reveals specific information about the structure and/or dynamics of the nucleic acid. Currently, there is no simple approach for making these data publically available in a standardized format. We therefore developed a standard for reporting the results of single nucleotide resolution nucleic acid structure mapping experiments, or SNRNASMs. We propose a schema for sharing nucleic acid chemical probing data that uses generic public servers for storing, retrieving, and searching the data. We have also developed a consistent nomenclature (ontology) within the Ontology of Biomedical Investigations (OBI), which provides unique identifiers (termed persistent URLs, or PURLs) for classifying the data. Links to standardized data sets shared using our proposed format along with a tutorial and links to templates can be found at http://snrnasm.bio.unc.edu.

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 (Selective 2′-Hydroxyl Acylation analyzed by Protection from Exoribonuclease) 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 E. 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.

Chemical acylation experiments showed that the RNA major groove, often assumed to be too deep and narrow to permit recognition interactions, is accessible at duplex termini. Reactivity extended further into the helix in the 5' than in the 3' direction. Asymmetric and large loops between helices uncoupled them, which yielded both enhanced reactivity at terminal base pairs and weaker stabilization enthalpy compared to that in small loops or symmetric loops of the same size. Uncoupled helices have effective helix ends with accessible major grooves; such motifs are attractive contributors to protein recognition, tertiary folding, and catalysis.

Most large ribozymes require protein cofactors in order to function efficiently. The yeast mitochondrial bI3 group I intron requires two proteins for efficient splicing, Mrs1 and the bI3 maturase. Mrs1 has evolved from DNA junction resolvases to function as an RNA cofactor for at least two group I introns; however, the RNA binding site and the mechanism by which Mrs1 facilitates splicing were unknown. Here we use high-throughput RNA structure analysis to show that Mrs1 binds a ubiquitous RNA tertiary structure motif, the GNRA tetraloop-receptor interaction, at two sites in the bI3 RNA. Mrs1 also interacts at similar tetraloop-receptor elements, as well as other structures, in the self-folding Azoarcus group I intron and in the RNase P enzyme. Thus, Mrs1 recognizes general features found in the tetraloop-receptor motif. Identification of the two Mrs1 binding sites now makes it possible to create a model of the complete six-component bI3 ribonucleoprotein. All protein cofactors bind at the periphery of the RNA such that every long-range RNA tertiary interaction is stabilized by protein binding, involving either Mrs1 or the bI3 maturase. This work emphasizes the strong evolutionary pressure to bolster RNA tertiary structure with RNA-binding interactions as seen in the ribosome, spliceosome, and other large RNA machines.

A striking and widespread observation is that higher-order folding for many RNAs is very slow, often requiring minutes. In some cases, slow folding reflects the need to disrupt stable, but incorrect, interactions. However, a molecular explanation for slow folding in most RNAs is unknown. The specificity domain of the Bacillus subtilis RNase P ribozyme undergoes a rate-limiting folding step on the minute time-scale. This RNA also contains a C2′-endo nucleotide at A130 that exhibits extremely slow local conformational dynamics. This nucleotide is evolutionarily conserved and essential for tRNA recognition by RNase P. Here we show that deleting this single nucleotide accelerates folding by an order of magnitude even though this mutation does not change the global fold of the RNA. These results demonstrate that formation of a single stacking interaction at a C2′-endo nucleotide comprises the rate-determining step for folding an entire 154 nucleotide RNA. C2′-endo nucleotides exhibit slow local dynamics in structures spanning isolated helices to complex tertiary interactions. Because the motif is both simple and ubiquitous, C2′-endo nucleotides may function as molecular timers in many RNA folding and ligand recognition reactions.

The difficulty of analyzing higher order RNA structure, especially for folding intermediates and for RNAs whose functions require domains that are conformationally flexible, emphasizes the need for new approaches for modeling RNA tertiary structure accurately. Here, we report a concise approach that makes use of facile RNA structure probing experiments that are then interpreted using a computational algorithm, carefully tailored to optimize both the resolution and refinement speed for the resulting structures, without requiring user intervention. The RNA secondary structure is first established using SHAPE chemistry. We then use a sequence-directed cleavage agent, which can be placed arbitrarily in many helical motifs, to obtain high quality inter-residue distances. We interpret this in-solution chemical information using a fast, coarse grained, discrete molecular dynamics engine in which each RNA nucleotide is represented by pseudoatoms for the phosphate, ribose, and nucleobase groups. By this approach, we refine base paired positions in yeast tRNA(Asp) to 4 A rmsd without any preexisting information or assumptions about secondary or tertiary structures. This blended experimental and computational approach has the potential to yield native-like models for the diverse universe of functionally important RNAs whose structures cannot be characterized by conventional structural methods.

Replication of human immunodeficiency virus requires binding of the viral Tat protein to its RNA target sequence TAR; peptides derived from Tat bind to a TAR "contact site" spanning 5 bp and a trinucleotide pyrimidine bulge. We find that high affinity binding requires a U residue in the bulge loop and 2 specific adjacent base pairs. Other bulged RNAs bind in a lower affinity nonspecific manner; sequence-specific binding requires a bulge loop of more than 1 nucleotide. Reaction with diethyl pyrocarbonate indicates that one effect of the bulge is to make the otherwise deep and narrow RNA major groove accessible. A model consistent with these data involves local distortion of A-form geometry at the bulge, which bends the helix and permits protein binding and interactive access in the RNA major groove.

Replication and pathogenesis of the human immunodeficiency virus (HIV) is tightly linked to the structure of its RNA genome, but genome structure in infectious virions is poorly understood. We invent high-throughput SHAPE (selective 2′-hydroxyl acylation analyzed by primer extension) technology, which uses many of the same tools as DNA sequencing, to quantify RNA backbone flexibility at single-nucleotide resolution and from which robust structural information can be immediately derived. We analyze the structure of HIV-1 genomic RNA in four biologically instructive states, including the authentic viral genome inside native particles. Remarkably, given the large number of plausible local structures, the first 10% of the HIV-1 genome exists in a single, predominant conformation in all four states. We also discover that noncoding regions functioning in a regulatory role have significantly lower (p-value < 0.0001) SHAPE reactivities, and hence more structure, than do viral coding regions that function as the template for protein synthesis. By directly monitoring protein binding inside virions, we identify the RNA recognition motif for the viral nucleocapsid protein. Seven structurally homologous binding sites occur in a well-defined domain in the genome, consistent with a role in directing specific packaging of genomic RNA into nascent virions. In addition, we identify two distinct motifs that are targets for the duplex destabilizing activity of this same protein. The nucleocapsid protein destabilizes local HIV-1 RNA structure in ways likely to facilitate initial movement both of the retroviral reverse transcriptase from its tRNA primer and of the ribosome in coding regions. Each of the three nucleocapsid interaction motifs falls in a specific genome domain, indicating that local protein interactions can be organized by the long-range architecture of an RNA. High-throughput SHAPE reveals a comprehensive view of HIV-1 RNA genome structure, and further application of this technology will make possible newly informative analysis of any RNA in a cellular transcriptome., Author Summary The function of the RNA genome of the human immunodeficiency virus (HIV) is determined both by its sequence and by its ability to fold back on itself to form specific higher-order structures. In order to describe physical structures in a region of the HIV RNA genome known to play multiple, critical roles in viral replication and pathogenesis, we invent a high-throughput, quantitative, and comprehensive structure-mapping approach that locates flexible (unpaired) nucleotides within a folded RNA, assaying hundreds of nucleotides at a time. We find that the first 10% of the HIV-1 genome has a single predominant structure and that regulatory motifs have significantly greater structure than do protein-coding segments. The HIV genome interacts with numerous proteins, including multiple copies of the nucleocapsid protein. We directly map RNA–protein interactions inside virions and discover that the nucleocapsid prottein interacts with viral RNA in at least three distinct ways, depending on the context within the overall genome structure. Further application of the high-throughput RNA-structure analysis tools described here will make it possible to address diverse structure–function relationships in intact cellular and viral RNAs., Development of novel, quantitative, high-throughput RNA structure analysis tools allows the outline of structure-function relationships for the first 10% of an HIV genome, discovery of structural differences between regulatory and coding regions, and analysis of protein-RNA interactions inside authentic virions.

RNA conformation is both highly dependent on and sensitive to the presence of charged ligands. Mono- and divalent ions stabilize the native fold of RNA, whereas other polyvalent cationic ligands can act to both stabilize or disrupt native RNA structure. In this work, we analyze the effects of two ligands, Mg (2+) and tobramycin, on the folding of S. cerevisiae tRNA (Asp) transcripts using single nucleotide resolution SHAPE chemistry. Surprisingly, reducing the Mg (2+) concentration favors a structural rearrangement in which the D- and variable loops pair. The tobramycin polycation binds to loops in tRNA (Asp) and induces RNA unfolding in two distinct transitions: the loss of tertiary interactions between the T- and D-loops followed by complete unfolding of the D-stem. Although Mg (2+) and tobramycin are relatively simple ligands, both modulate tRNA (Asp) folding in unanticipatedly complex ways, neither of which is consistent with simple hierarchical folding or unfolding of this RNA. Monitoring the structural consequences of ligand binding to RNA at single nucleotide resolution makes it possible to define intermediate structures that contribute to the complex energy landscapes often observed for RNA folding processes and lays the groundwork for a significantly improved understanding of the interactions between RNA and its solution environment.

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-1NL4-3, 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., Author Summary Human immunodeficiency virus (HIV) is a persistent and critical threat to human health. Replication and pathogenesis of HIV is governed by information encoded in its single-stranded RNA genome. In addition to coding for viral proteins, the HIV genomic RNA forms base paired and higher-order structures that are critical for viral replication. It is likely that only a subset of functional RNA motifs has been identified. Here, we interrogate the structures of three diverse HIV-related viral genomes by nucleotide-resolution chemical probing. The three genomes include HIV-1, the virus that infects humans, and SIVcpz and SIVmac, which are progenitors for the main branches of the two HIV evolutionary groups. We used a structure-informed alignment approach to generate consensus models for base-paired secondary structures that are shared by these three HIV-related genomes. With this approach, we were able to recapitulate all known RNA structures and, additionally, discovered multiple previously undescribed structural elements that are clearly conserved among major HIV groups. We anticipate that the methods described here will be broadly useful for RNA structure motif discovery and, more immediately, for identification of RNA targets in HIV that are promising sites for therapeutic intervention.

Intermediate states play well established roles in the folding and misfolding reactions of individual RNA and protein molecules. In contrast, the roles of transient structural intermediates in multi-component ribonucleoprotein (RNP) assembly processes and their potential for misassembly are largely unexplored. The mammalian signal recognition particle SRP19 protein is unstructured but forms a compact core domain and two extended RNA-binding loops upon binding the SRP RNA. The SRP54 protein subsequently binds to the fully assembled SRP19-RNA complex to form an intimate three-fold interface with both SRP19 and the SRP RNA and without significantly altering the structure of SRP19. We show, however, that the presence of SRP54 during SRP19-SRP RNA assembly dramatically alters the folding energy landscape to create a non-native folding pathway that leads to an aberrant SRP19-RNA conformation. The misassembled complex arises from the surprising ability of SRP54 to bind rapidly to an SRP19-RNA assembly intermediate and to interfere with subsequent folding of one of the SRP19 RNA-binding loops at the three-way protein-RNA interface. An incorrect temporal order of assembly thus readily yields a non-native three-component ribonucleoprotein particle. We propose there may exist a general requirement to regulate the order of interaction in multi-component RNP assembly by spatial or temporal compartmentalization of individual constituents in the cell.

Retroviruses selectively package two copies of their RNA genomes in the context of a large excess of nongenomic RNA. Specific packaging of genomic RNA is achieved, in part, by recognizing RNAs that form a poorly understood dimeric structure at their 5′ ends. We identify, quantify the stability of, and use extensive experimental constraints to calculate a 3D model for a tertiary structure domain that mediates specific interactions between RNA genomes in a gamma retrovirus. In an initial interaction, two stem–loop structures from one RNA form highly stringent cross-strand loop–loop base pairs with the same structures on a second genomic RNA. Upon subsequent folding to the final dimer state, these intergenomic RNA interactions convert to a high affinity and compact tertiary structure, stabilized by interdigitated interactions between U-shaped RNA units. This retroviral conformational switch model illustrates how two-step formation of an RNA tertiary structure yields a stringent molecular recognition event at early assembly steps that can be converted to the stable RNA architecture likely packaged into nascent virions.

Ribose 2'-amine substitutions are broadly useful as structural probes in nucleic acids. In addition, structure-selective chemical reaction at 2'-amine groups is a robust technology for interrogating local nucleotide flexibility and conformational changes in RNA and DNA. We analyzed crystal structures for several RNA duplexes containing 2'-amino cytidine (C(N)) residues that form either C(N)-G base pairs or C(N)-A mismatches. The 2'-amine substitution is readily accommodated in an A-form RNA helix and thus differs from the C2'-endo conformation observed for free nucleosides. The 2'-amide product structure was visualized directly by acylating a C(N)-A mismatch in intact crystals and is also compatible with A-form geometry. To visualize conformations able to facilitate formation of the amide-forming transition state, in which the amine nucleophile carries a positive partial charge, we analyzed crystals of the C(N)-A duplex at pH 5, where the 2'-amine is protonated. The protonated amine moves to form a strong electrostatic interaction with the 3'-phosphodiester. Taken together with solution-phase experiments, 2'-amine acylation is likely facilitated by either of two transition states, both involving precise positioning of the adjacent 3'-phosphodiester group.

Ligation of an adapter oligonucleotide to a single-stranded cDNA is central to many molecular biology techniques. Current single-stranded ligation approaches suffer from low efficiencies and are strongly inhibited by preexisting DNA secondary structure. We develop an approach for ligating low concentrations of single-stranded DNAs to a DNA adapter with near-quantitative efficiency, unaffected by secondary structure in the target DNA. This efficient DNA ligation reaction will facilitate development of robust procedures for quantifying small amounts of highly structured cDNAs and their RNA templates.

Native polyacrylamide gel electrophoresis is a powerful approach for visualizing RNA folding states and folding intermediates. Tris-borate has a high-buffering capacity and is therefore widely used in electrophoresis-based investigations of RNA structure and folding. However, the effectiveness of Tris-borate as a counterion for RNA has not been systematically investigated. In a recirculated Hepes/KCl buffer, the catalytic core of the bI5 group I intron RNA undergoes a conformational collapse characterized by a bulk transition midpoint, or Mg1/2, of approximately 3 mM, consistent with extensive independent biochemical experiments. In contrast, in Tris-borate, RNA collapse has a much smaller apparent Mg1/2, equal to 0.1 mM, because in this buffer the RNA undergoes a different, large amplitude, folding transition at low Mg2+ concentrations. Analysis of structural neighbors using a short-lived, RNA-tethered, photocrosslinker indicates that the global RNA structure eventually converges in the two buffer systems, as the divalent ion concentration approaches approximately 1 mM Mg2+. The weak capacity of Tris-borate to stabilize RNA folding may reflect relatively unfavorable interactions between the bulky Tris-borate ion and RNA or partial coordination of RNA functional groups by borate. Under some conditions, Tris-borate is a poor counterion for RNA and its use merits careful evaluation in RNA folding studies.

Prior to folding to a native functional structure, many large RNAs form conformationally collapsed states. Formation of the near-native collapsed state for the bI5 group I intron RNA plays an obligatory role in self-chaperoning assembly with its CBP2 protein cofactor by preventing formation of stable, misassembled complexes. We show that the collapsed state is essential because CBP2 assembles indiscriminately with the bI5 RNA in any folding state to form long-lived complexes. The most stable protein interaction site in the expanded state-CBP2 complex overlaps, but is not identical to, the native site. Folding to the collapsed state circumvents two distinct misassembly events: inhibitory binding by multiple equivalents of CBP2 and formation of bridged complexes in which CBP2 straddles cognate and noncognate RNAs. Strikingly, protein-bound sites in the expanded state RNA complex are almost the inverse of native RNA-RNA and RNA-protein interactions, indicating that folding to the collapsed state significantly reduces the fraction of RNA surfaces accessible for misassembly. The self-chaperoning function for the bI5 collapsed state is likely to be conserved in other ribonucleoproteins where a protein cofactor binds tightly at a simple RNA substructure or has an RNA binding surface composed of multiple functional sites.

Many large RNAs form conformationally collapsed, but non-native, states prior to folding to the native state or assembling with protein cofactors. Although RNA collapsed states play fundamental roles in RNA folding and ribonucleoprotein assembly processes, their structures have been poorly understood. We obtained 12 high-quality structural constraints for the collapsed state formed by the catalytic core of the bI5 intron RNA using site-specific cross-linking mediated by a short-lived reactant. RNA tertiary structures in the collapsed and native states are indistinguishable, even though only the native state forms a solvent-inaccessible core. Thus, structural neighbors in the collapsed state, including several long-range tertiary interactions, are approximately as close in space as in the native state, but RNA packing is sufficiently loose or dynamic to allow access by solvent. Binding by the obligate CBP2 protein cofactor has almost no effect on structural neighbors reported by cross-linking, even though protein binding chases the RNA from the collapsed state to the native state. Protein binding thus appears to promote only the final few angstroms of RNA folding rather than mediate global conformational rearrangements in the catalytic core. The bI5 RNA collapsed state functions to self-chaperone ribonucleoprotein assembly because this conformationally restrained structure lies very near that of the native state and excludes structures that otherwise misassemble efficiently.