Analyzing RNA posttranscriptional modifications to decipher the epitranscriptomic code.

The discovery of RNA silencing has revealed that non-protein-coding sequences (ncRNAs) can cover essential roles in regulatory networks and their malfunction may result in severe consequences on human health. These findings have prompted a general reassessment of the significance of RNA as a key player in cellular processes. This reassessment, however, will not be complete without a greater understanding of the distribution and function of the over 170 variants of the canonical ribonucleotides, which contribute to the breathtaking structural diversity of natural RNA. This review surveys the analytical approaches employed for the identification, characterization, and detection of RNA posttranscriptional modifications (rPTMs). The merits of analyzing individual units after exhaustive hydrolysis of the initial biopolymer are outlined together with those of identifying their position in the sequence of parent strands. Approaches based on next generation sequencing and mass spectrometry technologies are covered in depth to provide a comprehensive view of their respective merits. Deciphering the epitranscriptomic code will require not only mapping the location of rPTMs in the various classes of RNAs, but also assessing the variations of expression levels under different experimental conditions. The fact that no individual platform is currently capable of meeting all such demands implies that it will be essential to capitalize on complementary approaches to obtain the desired information. For this reason, the review strived to cover the broadest possible range of techniques to provide readers with the fundamental elements necessary to make informed choices and design the most effective possible strategy to accomplish the task at hand.

Agnostic Framework for the Classification/Identification of Organisms Based on RNA Post-Transcriptional Modifications.

We propose a novel approach for building a classification/identification framework based on the full complement of RNA post-transcriptional modifications (rPTMs) expressed by an organism at basal conditions. The approach relies on advanced mass spectrometry techniques to characterize the products of exonuclease digestion of total RNA extracts. Sample profiles comprising identities and relative abundances of all detected rPTM were used to train and test the capabilities of different machine learning (ML) algorithms. Each algorithm proved capable of identifying rigorous decision rules for differentiating closely related classes and correctly assigning unlabeled samples. The ML classifiers resolved different members of the Enterobacteriaceae family, alternative Escherichia coli serotypes, a series of Saccharomyces cerevisiae knockout mutants, and primary cells of the Homo sapiens central nervous system, which shared very similar genetic backgrounds. The excellent levels of accuracy and resolving power achieved by training on a limited number of classes were successfully replicated when the number of classes was significantly increased to escalate complexity. A dendrogram generated from ML-curated data exhibited a hierarchical organization that closely resembled those afforded by established taxonomic systems. Finer clustering patterns revealed the extensive effects induced by the deletion of a single pivotal gene. This information provided a putative roadmap for exploring the roles of rPTMs in their respective regulatory networks, which will be essential to decipher the epitranscriptomics code. The ubiquitous presence of RNA in virtually all living organisms promises to enable the broadest possible range of applications, with significant implications in the diagnosis of RNA-related diseases.

Positive-sense RNA viruses reveal the complexity and dynamics of the cellular and viral epitranscriptomes during infection.

More than 140 post-transcriptional modifications (PTMs) are known to decorate cellular RNAs, but their incidence, identity and significance in viral RNA are still largely unknown. We have developed an agnostic analytical approach to comprehensively survey PTMs on viral and cellular RNAs. Specifically, we used mass spectrometry to analyze PTMs on total RNA isolated from cells infected with Zika virus, Dengue virus, hepatitis C virus (HCV), poliovirus and human immunodeficiency virus type 1. All five RNA viruses significantly altered global PTM landscapes. Examination of PTM profiles of individual viral genomes isolated by affinity capture revealed a plethora of PTMs on viral RNAs, which far exceeds the handful of well-characterized modifications. Direct comparison of viral epitranscriptomes identified common and virus-specific PTMs. In particular, specific dimethylcytosine modifications were only present in total RNA from virus-infected cells, and in intracellular HCV RNA, and viral RNA from Zika and HCV virions. Moreover, dimethylcytosine abundance during viral infection was modulated by the cellular DEAD-box RNA helicase DDX6. By opening the Pandora's box on viral PTMs, this report presents numerous questions and hypotheses on PTM function and strongly supports PTMs as a new tier of regulation by which RNA viruses subvert the host and evade cellular surveillance systems.

Bifunctional cross-linking approaches for mass spectrometry-based investigation of nucleic acids and protein-nucleic acid assemblies.

With the goal of expanding the very limited toolkit of cross-linking agents available for nucleic acids and their protein complexes, we evaluated the merits of a wide range of bifunctional agents that may be capable of reacting with the functional groups characteristic of these types of biopolymers. The survey specifically focused on the ability of test reagents to produce desirable inter-molecular conjugates, which could reveal the identity of interacting components and the position of mutual contacts, while also considering a series of practical criteria for their utilization as viable nucleic acid probes. The survey employed models consisting of DNA, RNA, and corresponding protein complexes to mimic as close as possible typical applications. Denaturing polyacrylamide gel electrophoresis (PAGE) and mass spectrometric (MS) analyses were implemented in concert to monitor the formation of the desired conjugates. In particular, the former was used as a rapid and inexpensive tool for the efficient evaluation of cross-linker activity under a broad range of experimental conditions. The latter was applied after preliminary rounds of reaction optimization to enable full-fledged product characterization and, more significantly, differentiation between mono-functional and intra- versus inter-molecular conjugates. This information provided the feedback necessary to further optimize reaction conditions and explain possible outcomes. Among the reagents tested in the study, platinum complexes and nitrogen mustards manifested the most favorable characteristics for practical cross-linking applications, whereas other compounds provided inferior yields, or produced rather unstable conjugates that did not survive the selected analytical conditions. The observed outcomes will help guide the selection of the most appropriate cross-linking reagent for a specific task, whereas the experimental conditions described here will provide an excellent starting point for approaching these types of applications. As a whole, the results of the survey clearly emphasize that finding a universal reagent, which may afford excellent performance with all types of nucleic acid substrates, will require extending the exploration beyond the traditional chemistries employed to modify the constitutive functional groups of these vital biopolymers.

Submicrometer Nanospray Emitters Provide New Insights into the Mechanism of Cation Adduction to Anionic Oligonucleotides.

The electrospray-MS analysis of oligonucleotides is hampered by nonvolatile metal cations, which may produce adducts responsible for signal suppression and loss of resolution. Alternative to replacing metal cations with MS-friendly ammonium, we explored the utilization of nanospray emitters with submicrometer-diameter tips, which was shown to benefit the analysis of protein samples containing elevated salt concentrations. We demonstrated that such benefits are not limited to proteins, but extend also to oligonucleotide samples analyzed in the negative ion mode. At elevated Na+/Mg2+ concentrations, submicrometer tips produced significantly greater signal-to-noise ratios, as well as greatly reduced adducts and salt clusters, than observed when utilizing micrometer tips. These effects were marginally affected by emitter composition (i.e., borosilicate versus quartz), but varied according to salt concentration and number of oligonucleotide phosphates. The results confirmed that adduct formation is driven by the concentrating effects of the desolvation process, which leads to greatly increased solute concentrations as the volume of the droplet decreases. The process promotes cation-phosphate interactions that may not have necessarily existed in the initial sample, but nevertheless shape the observed adduct series. Therefore, such series may not accurately reflect the distribution of counterions surrounding the analyte in solution. No adverse effects were noted on specific metal interactions, such as those present in a model drug-DNA assembly. These observations indicate that the utilization of submicrometer tips represents an excellent alternative to traditional ammonium-replacement approaches, which enables the analysis of oligonucleotides in the presence of Na+/Mg2+ concentrations capable of preserving their structure and functional properties.

Global Epitranscriptomics Profiling of RNA Post-Transcriptional Modifications as an Effective Tool for Investigating the Epitranscriptomics of Stress Response.

The simultaneous detection of all the post-transcriptional modifications (PTMs) that decorate cellular RNA can provide comprehensive information on the effects of changing environmental conditions on the entire epitranscriptome. To capture this type of information, we performed the analysis of ribonucleotide mixtures produced by hydrolysis of total RNA extracts from S. cerevisiae that was grown under hyperosmotic and heat shock conditions. Their global PTM profiles clearly indicated that the cellular responses to these types of stresses involved profound changes in the production of specific PTMs. The observed changes involved not only up-/down-regulation of typical PTMs, but also the outright induction of new ones that were absent under normal conditions, or the elimination of others that were normally present. Pointing toward the broad involvement of different classes of RNAs, many of the newly observed PTMs differed from those engaged in the known tRNA-based mechanism of translational recoding, which is induced by oxidative stress. Some of the expression effects were stress-specific, whereas others were not, thus suggesting that RNA PTMs may perform multifaceted activities in stress response, which are subjected to distinctive regulatory pathways. To explore their signaling networks, we implemented a strategy based on the systematic deletion of genes that connect established response genes with PTM biogenetic enzymes in a putative interactomic map. The results clearly identified PTMs that were under direct HOG control, a well-known protein kinase pathway involved in stress response in eukaryotes. Activation of this signaling pathway has been shown to result in the stabilization of numerous mRNAs and the induction of selected lncRNAs involved in chromatin remodeling. The fact that PTMs are capable of altering the activity of the parent RNAs suggest their possible participation in feedback mechanisms aimed at modulating the regulatory functions of such RNAs. This tantalizing hypothesis will be the object of future studies.

Profiling ribonucleotide modifications at full-transcriptome level: a step toward MS-based epitranscriptomics.

The elucidation of the biological significance of RNA post-transcriptional modifications is hampered by the dearth of effective high-throughput sequencing approaches for detecting, locating, and tracking their levels as a function of predetermined experimental factors. With the goal of confronting this knowledge gap, we devised a strategy for completing global surveys of all ribonucleotide modifications in a cell, which is based on the analysis of whole cell extracts by direct infusion electrospray ionization mass spectrometry (ESI-MS). Our approach eschews chromatographic separation to promote instead the direct application of MS techniques capable of providing detection, differentiation, and quantification of post-transcriptional modifications (PTMs) in complex ribonucleotide mixtures. Accurate mass analysis was used to carry out database-aided identification of PTMs, whereas multistep tandem mass spectrometry (MS(n)) and consecutive reaction monitoring (CRM) provided the necessary structural corroboration. We demonstrated that heat-map plots afforded by ion mobility spectrometry mass spectrometry (IMS-MS) can provide comprehensive modification profiles that are unique for different cell types and metabolic states. We showed that isolated tRNA samples can be used as controlled sources of PTMs in standard-additions quantification. Intrinsic internal standards enable direct comparisons of heat-maps obtained under different experimental conditions, thus offering the opportunity to evaluate the global effects of such conditions on the expression levels of all PTMs simultaneously. This type of comparative analysis will be expected to support the investigation of the system biology of RNA modifications, which will be aimed at exploring mutual correlations of their expression levels and providing new valuable insights into their biological significance.

A2T and A2V Aß peptides exhibit different aggregation kinetics, primary nucleation, morphology, structure, and LTP inhibition.

The histopathological hallmark of Alzheimer's disease (AD) is the aggregation and accumulation of the amyloid beta peptide (Aß) into misfolded oligomers and fibrils. Here we examine the biophysical properties of a protective Aß variant against AD, A2T, and a causative mutation, A2T, along with the wild type (WT) peptide. The main finding here is that the A2V native monomer is more stable than both A2T and WT, and this manifests itself in different biophysical behaviors: the kinetics of aggregation, the initial monomer conversion to an aggregation prone state (primary nucleation), the abundances of oligomers, and extended conformations. Aggregation reaction modeling of the conversion kinetics from native monomers to fibrils predicts the enhanced stability of the A2V monomer, while ion mobility spectrometry-mass spectrometry measures this directly confirming earlier predictions. Additionally, unique morphologies of the A2T aggregates are observed using atomic force microscopy, providing a basis for the reduction in long term potentiation inhibition of hippocampal cells for A2T compared with A2V and the wild type (WT) peptide. The stability difference of the A2V monomer and the difference in aggregate morphology for A2T (both compared with WT) are offered as alternate explanations for their pathological effects.

Modular calibrant sets for the structural analysis of nucleic acids by ion mobility spectrometry mass spectrometry.

This study explored the use of modular nucleic acid (NA) standards to generate calibration curves capable of translating primary ion mobility readouts into corresponding collision cross section (CCS) data. Putative calibrants consisted of single- (ss) and double-stranded (ds) oligo-deoxynucleotides reaching up to ∼40 kDa in size (i.e., 64 bp) and ∼5700 Å(2) in CCS. To ensure self-consistency among reference CCS values, computational data obtained in house were preferred to any experimental or computational data from disparate sources. Such values were obtained by molecular dynamics (MD) simulations and either the exact hard sphere scattering (EHSS) or the projection superposition approximation (PSA) methods, and then plotted against the corresponding experimental values to generate separate calibration curves. Their performance was evaluated on the basis of their correlation coefficients and ability to provide values that matched the CCS of selected test samples mimicking typical unknowns. The results indicated that the predictive power benefited from the exclusion of higher charged species that were more susceptible to the destabilizing effects of Coulombic repulsion. The results revealed discrepancies between EHSS and PSA data that were ascribable to the different approximations used to describe the ion mobility process. Within the boundaries defined by these approximations and the challenges of modeling NA structure in a solvent-free environment, the calibrant sets enabled the experimental determination of CCS with excellent reproducibility (precision) and error (accuracy), which will support the analysis of progressively larger NA samples of biological significance.

Structures of the kinetically trapped i-motif DNA intermediates.

In the present work, the conformational dynamics and folding pathways of i-motif DNA were studied in solution and in the gas-phase as a function of the solution pH conditions using circular dichroism (CD), photoacoustic calorimetry analysis (PAC), trapped ion mobility spectrometry-mass spectrometry (TIMS-MS), and molecular dynamics (MD). Solution studies showed at thermodynamic equilibrium the existence of a two-state folding mechanism, whereas during the pH = 7.0 → 4.5 transition a fast and slow phase (ΔHfast + ΔHslow = 43 ± 7 kcal mol-1) with a volume change associated with the formation of hemiprotonated cytosine base pairs and concomitant collapse of the i-motif oligonucleotide into a compact conformation were observed. TIMS-MS experiments showed that gas-phase, kinetically trapped i-motif DNA intermediates produced by nanoESI are preserved, with relative abundances depending on the solution pH conditions. In particular, a folded i-motif DNA structure was observed in nanoESI-TIMS-MS for low charge states in both positive and negative ion mode (e.g., z = ±3 to ±5) at low pH conditions. As solution pH increases, the cytosine neutralization leads to the loss of cytosine-cytosine+ (C·CH+) base pairing in the CCC strands and in those conditions we observe partially unfolded i-motif DNA conformations in nanoESI-TIMS-MS for higher charge states (e.g., z = -6 to -9). Collisional induced activation prior to TIMS-MS showed the existence of multiple local free energy minima, associated with the i-motif DNA unfolding at z = -6 charge state. For the first time, candidate gas-phase structures are proposed based on mobility measurements of the i-motif DNA unfolding pathway. Moreover, the inspection of partially unfolded i-motif DNA structures (z = -7 and z = -8 charge states) showed that the presence of inner cations may or may not induce conformational changes in the gas-phase. For example, incorporation of ammonium adducts does not lead to major conformational changes while sodium adducts may lead to the formation of sodium mediated bonds between two negatively charged sides inducing the stabilization towards more compact structures in new local, free energy minima in the gas-phase.

Investigating the Merits of Microfluidic Capillary Zone Electrophoresis-Mass Spectrometry (CZE-MS) in the Bottom-Up Characterization of Larger RNAs.

Established bottom-up approaches for the characterization of nucleic acids (NAs) rely on the strand-cleavage activity of nucleotide-specific endonucleases to generate smaller oligonucleotides amenable to gas-phase sequencing. The complexity of these hydrolytic mixtures calls for the utilization of a front-end separation to facilitate full mass spectrometric (MS) characterization. This report explored the merits of microfluidic capillary zone electrophoresis (CZE) as a possible alternative to common liquid chromatography techniques. An oligonucleotide ladder was initially employed to investigate the roles of fundamental analyte features and experimental parameters in determining the outcome of CZE-MS analyses. The results demonstrated the ability to fully resolve the various rungs into discrete electrophoretic peaks with full-width half-height (FWHH) resolution that was visibly affected by the overall amount of material injected into the system. Analogous results were obtained from a digestion mixture prepared by treating yeast tRNAPhe (75 nt) with RNase T1, which provided several well-resolved peaks in spite of the increasing sample heterogeneity. The regular shapes of such peaks, however, belied the fact that most of them contained sets of comigrating species, as shown by the corresponding MS spectra. Even though it was not possible to segregate each species into an individual electrophoretic peak, the analysis still proved capable of unambiguously identifying a total of 29 hydrolytic products, which were sufficient to cover 96% of the tRNAPhe's sequence. Their masses accurately reflected the presence of modified nucleotides characteristic of this type of substrate. The analysis of a digestion mixture obtained from the 364 nt HIV-1 5'-UTR proved to be more challenging. The electropherogram displayed fewer well-resolved peaks and significantly greater incidence of product comigration. In this case, fractionating the highly heterogeneous mixture into discrete bands helped reduce signal suppression and detection bias. As a result, the corresponding MS data enabled the assignment of 248 products out of the possible 513 predicted from the 5'-UTR sequence, which afforded 100% sequence coverage. This figure represented a significant improvement over the 36 total products identified earlier under suboptimal conditions, which afforded only 57% coverage, or the 83 observed by direct infusion nanospray-MS (72%). These results provided a measure of the excellent potential of the technique to support the bottom-up characterization of progressively larger NA samples, such as putative NA therapeutics and mRNA vaccines.

The essential role of stacking adenines in a two-base-pair RNA kissing complex.

In minimal RNA kissing complexes formed between hairpins with cognate GACG tetraloops, the two tertiary GC pairs are likely stabilized by the stacking of 5'-unpaired adenines at each end of the short helix. To test this hypothesis, we mutated the flanking adenines to various nucleosides and examined their effects on the kissing interaction. Electrospray ionization mass spectrometry was used to detect kissing dimers in a multiequilibria mixture, whereas optical tweezers were applied to monitor the (un)folding trajectories of single RNA molecules. The experimental findings were rationalized by molecular dynamics simulations. Together, the results showed that the stacked adenines are indispensable for the tertiary interaction. By shielding the tertiary base pairs from solvent and reducing their fraying, the stacked adenines made terminal pairs act more like interior base pairs. The purine double-ring of adenine was essential for effective stacking, whereas additional functional groups modulated the stabilizing effects through varying hydrophobic and electrostatic forces. Furthermore, formation of the kissing complex was dominated by base pairing, whereas its dissociation was significantly influenced by the flanking bases. Together, these findings indicate that unpaired flanking nucleotides play essential roles in the formation of otherwise unstable two-base-pair RNA tertiary interactions.

Targeting loop adenines in G-quadruplex by a selective oxirane.

Caught in the oxirane: Naphthalene diimides conjugated to a quinone methide and an oxirane have been synthesized and investigated as selective DNA G-quadruplex alkylating agents. The oxirane derivative generates a stable adduct with a G-quadruplex and shows selective alkylation of the loop adenines, as illustrated.

The RNA Modification Database, RNAMDB: 2011 update.

Since its inception in 1994, The RNA Modification Database (RNAMDB, http://rna-mdb.cas.albany.edu/RNAmods/) has served as a focal point for information pertaining to naturally occurring RNA modifications. In its current state, the database employs an easy-to-use, searchable interface for obtaining detailed data on the 109 currently known RNA modifications. Each entry provides the chemical structure, common name and symbol, elemental composition and mass, CA registry numbers and index name, phylogenetic source, type of RNA species in which it is found, and references to the first reported structure determination and synthesis. Though newly transferred in its entirety to The RNA Institute, the RNAMDB continues to grow with two notable additions, agmatidine and 8-methyladenosine, appended in the last year. The RNA Modification Database is staying up-to-date with significant improvements being prepared for inclusion within the next year and the following year. The expanded future role of The RNA Modification Database will be to serve as a primary information portal for researchers across the entire spectrum of RNA-related research.

Integrating Internal Fragments in the Interpretation of Top-Down Sequencing Data of Larger Oligonucleotides.

In the context of direct top-down analysis or concerted bottom-up characterization of nucleic acid samples, the waning yield of terminal fragments as a function of precursor ion size poses a significant challenge to the gas-phase sequencing of progressively larger oligonucleotides. In this report, we examined the behavior of oligoribonucleotide samples ranging from 20 to 364 nt upon collision-induced dissociation (CID). The experimental data showed a progressive shift from terminal to internal fragments as a function of size. The systematic evaluation of experimental factors, such as collision energy, precursor charge, sample temperature, and the presence of chaotropic agents, showed that this trend could be modestly alleviated but not suppressed. This inexorable effect, which has been reported also for other activation techniques, prompted a re-examination of the features that have traditionally discouraged the utilization of internal fragments as a source of sequence information in data interpretation procedures. Our simulations highlighted the ability of internal fragments to produce self-consistent ladders with either end corresponding to each nucleotide in the sequence, which enables both proper alignment and correct recognition of intervening nucleotides. In turn, contiguous ladders display extensive overlaps with one another and with the ladders formed by terminal fragments, which unambiguously constrain their mutual placement within the analyte sequence. The experimental data borne out the predictions by showing ladders with extensive overlaps, which translated into uninterrupted "walks" covering the entire sequence with no gaps from end to end. More significantly, the results showed that combining the information afforded by internal and terminal ladders resulted in much a greater sequence coverage and nucleotide coverage depth than those achievable when either type of information was considered separately. The examination of a series of 58-mer oligonucleotides with high sequence homology showed that the assignment ambiguities engendered by internal fragments did not significantly exceed those afforded by the terminal ones. Therefore, the balance between potential benefits and perils of including the former makes a compelling argument for the development of integrated data interpretation strategies, which are better equipped for dealing with the changing fragmentation patterns obtained from progressively larger oligonucleotides.

Suppression of mutant C9orf72 expression by a potent mixed backbone antisense oligonucleotide.

Expansions of a G4C2 repeat in the C9ORF72 gene are the most common genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD), two devastating adult-onset neurodegenerative disorders. Using C9-ALS/FTD patient-derived cells and C9ORF72 BAC transgenic mice, we generated and optimized antisense oligonucleotides (ASOs) that selectively blunt expression of G4C2 repeat-containing transcripts and effectively suppress tissue levels of poly(GP) dipeptides. ASOs with reduced phosphorothioate content showed improved tolerability without sacrificing efficacy. In a single patient harboring mutant C9ORF72 with the G4C2 repeat expansion, repeated dosing by intrathecal delivery of the optimal ASO was well tolerated, leading to significant reductions in levels of cerebrospinal fluid poly(GP). This report provides insight into the effect of nucleic acid chemistry on toxicity and, to our knowledge, for the first time demonstrates the feasibility of clinical suppression of the C9ORF72 gene. Additional clinical trials will be required to demonstrate safety and efficacy of this therapy in patients with C9ORF72 gene mutations.

SHAMS: combining chemical modification of RNA with mass spectrometry to examine polypurine tract-containing RNA/DNA hybrids.

Selective 2'-hydroxyl acylation analyzed by primer extension (SHAPE) has gained popularity as a facile method of examining RNA structure both in vitro and in vivo, exploiting accessibility of the ribose 2'-OH to acylation by N-methylisatoic anhydride (NMIA) in unpaired or flexible configurations. Subsequent primer extension terminates at the site of chemical modification, and these products are fractionated by high-resolution gel electrophoresis. When applying SHAPE to investigate structural features associated with the wild-type and analog-substituted polypurine tract (PPT)-containing RNA/DNA hybrids, their size (20-25 base pairs) rendered primer extension impractical. As an alternative method of detection, we reasoned that chemical modification could be combined with tandem mass spectrometry, relying on the mass increment of RNA fragments containing the NMIA adduct (M(r) = 133 Da). Using this approach, we demonstrate both specific modification of the HIV-1 PPT RNA primer and variations in its acylation pattern induced by replacing template nucleotides with a non-hydrogen-bonding thymine isostere. Our selective 2'-hydroxyl acylation analyzed by mass spectrometry strategy (SHAMS) should find utility when examining the structure of small RNA fragments or RNA/DNA hybrids where primer extension cannot be performed.

MS3D structural elucidation of the HIV-1 packaging signal.

The structure of HIV-1 Psi-RNA has been elucidated by a concerted approach combining structural probes with mass spectrometric detection (MS3D), which is not affected by the size and crystallization properties of target biomolecules. Distance constraints from bifunctional cross-linkers provided the information required for assembling an all-atom model from the high-resolution coordinates of separate domains by triangulating their reciprocal placement in 3D space. The resulting structure revealed a compact cloverleaf morphology stabilized by a long-range tertiary interaction between the GNRA tetraloop of stemloop 4 (SL4) and the upper stem of stemloop 1 (SL1). The preservation of discrete stemloop structures ruled out the possibility that major rearrangements might produce a putative supersite with enhanced affinity for the nucleocapsid (NC) domain of the viral Gag polyprotein, which would drive genome recognition and packaging. The steric situation of single-stranded regions exposed on the cloverleaf structure offered a valid explanation for the stoichiometry exhibited by full-length Psi-RNA in the presence of NC. The participation of SL4 in a putative GNRA loop-receptor interaction provided further indications of the plasticity of this region of genomic RNA, which can also anneal with upstream sequences to stabilize alternative conformations of the 5' untranslated region (5'-UTR). Considering the ability to sustain specific NC binding, the multifaceted activities supported by the SL4 sequence suggest a mechanism by which Gag could actively participate in regulating the vital functions mediated by 5'-UTR. Substantiated by the 3D structure of Psi-RNA, the central role played by SL4 in specific RNA-RNA and protein-RNA interactions advances this domain as a primary target for possible therapeutic intervention.