Pedestrian route choice, the process by which individuals decide on their walking path between two locations, is a fundamental problem across disciplines. Because this behaviour is investigated from different conceptual and methodological angles, and because it strongly depends on the environmental context, it is challenging to establish a systematic framework for research. Here, by reviewing previous work, we identify four principles for pedestrian route choice that are relevant across disciplines. First, ‘information perception’ deals with how pedestrians can perceive information selectively and purposely, given the limited available information. Second, ‘information integration’ considers how pedestrians subjectively integrate environmental spatial information into mental representations. Third, ‘responding to information’ is concerned with how pedestrians tend to be attracted and repelled by specific attributes individually and how this can lead to positive or negative feedback loops across many individuals. Fourth ‘decision-making mechanisms' describe how pedestrians trade off the evidence provided by different attributes. How pedestrians perceive, integrate, respond to, and act upon information is not fixed but varies with the context. We give examples for each principle and explain how these principles shape pedestrian choice behaviours. We hope this contribution provides a systematic overview of the field and helps to spark inspiration among specialists. Fortunately, you have a map that precisely shows the correct information to get you where you would like to go. However, there are several routes, including direct and short ones, and scenic ones that are indirect and pass through narrow lanes. Which route will you take? Now imagine that you visit a museum for the first time when the fire alarm starts to sound. When you are looking for a way out, you probably do not consider how scenic the way is. Instead, you may be looking for evacuation signs and where others are going, but it may be dark, or worse, rooms may be filled with smoke. When all cues suggest the same route, it is not difficult to decide in these situations. If not, you will have to somehow trade off different route properties that you are aware of. While these two scenarios are probably not an everyday occurrence, pedestrian route choice is involved in many aspects of our daily lives, such as commuting, for example. We go to work on a specific route and go home on the same or another route. This route choice has already taken place many times, it may not be a conscious decision sometimes, and it may be determined by choice inertia. Regardless of the specific mechanisms for how we select routes, they shape our spatial behaviour.The above examples illustrate realistic route choices people may face. Route choice is the spatial choice pedestrians make between a set of alternatives with the goal of reaching the desired destination, a process people have to deal with on a daily basis or in emergencies ,2. It isa, the number of publications on this topic has been increasing substantially since the early 2000s and over 7000 studies have contributed to this research object. When reviewing work on pedestrian route choice, we found two main reasons that make it complex.Pedestrian route choice has long been a research topic and has drawn much attention in recent decades. As shown in figure 1b illustrates the percentage of all publications on this topic in different disciplines. For example, transportation scientists may develop route choice models calibrated on empirical data that involve interactions between pedestrians and vehicles with the goal of predicting future traffic conditions which in turn will support transportation control and planning in cities . S. Sb) [56b) . While tit usage .d, where pedestrians have to choose either a longer route or a shorter route to reach a destination. Suppose they are more likely to choose the less busy route to lower their individual travel cost, making this preferred route busier than the other route. However, as more people choose the shorter route, it becomes congested, which incurs a time delay, making the shorter route less and less attractive compared to the longer route. As a result, the process reaches an equilibrium where there is no longer an incentive to preferentially choose either of the routes (figure 4e) [Figure 4f illustrates another example where pedestrians are distributed unevenly in a room with four exits prior to the onset of egress from this room. If each pedestrian chooses an exit based on an objective estimation of the remaining travel time, then they will initially tend to choose the nearest exit. Over time, the increasing number of people clustered around exits will cause congestion and increase the estimated travel time for exits causing pedestrians to avoid exits, even if they are nearby. As a result, pedestrians will distribute approximately evenly across the available exits (figure 4g) [By contrast, negative feedback is a process when the results of an action inhibit that action from continuing to occur. It generally promotes stability and reduces the effects of perturbations. Consider, for example, a simple network, as shown in figure 4gure 4e) . Figure gure 4g) . This pr. 2.4Pedestrians perform trade-offs based on the evidence provided by different attributes.So far, we have discussed how pedestrians perceive information, how they integrate it into mental representations, and how some aspects of environments are desirable and others are undesirable in route choice. Now, we discuss the mechanisms by which pedestrians decide on their route. There is a consensus that if pedestrians are aware of several route options, they trade off the evidence provided by different environmental attributes associated with the alternatives when making their decision. The precise process for how this trade-off is arrived at is unknown. There are two broad theoretical paradigms that are both useful for understanding and predicting pedestrian route choice. The first can be described as utility theory. It assumes pedestrians assign a value to environmental characteristics and then choose the option with the best value, potentially subject to some uncertainty . The sec. 2.4.1In utility models, the preference of a pedestrian for each option is assigned a quantitative value, known as a utility. The utility measures the degree to which the goals of an individual are achieved as a result of their decisions. Thus, the utility assigned to environmental features, such as exit signs, or properties of routes, such as their length, is crucial. The choice set contains all available mutually exclusive alternatives which in the context of pedestrian route choice is a finite number of options .nU consists of a deterministic component nV and a random component ɛn. The former is calculated by combining the utilities of separate attributes associated with alternative n. One example for this is given in equation (2.2), where nV is expressed as a linear combination of the contribution of the k observed attributes, with a vector of utility parameters βk that captures the relative weight of the corresponding attributes. The random component ɛn can be interpreted to describe lack of information or other cognitive processes in pedestrians, or it can reflect our incomplete knowledge of the decision-making processes in pedestrians, such as not knowing all factors that influence pedestrian route choice or differences between individuals. Different assumptions about the distributions of the random utility component result in different utility models. For example, in probit models, logit models and multinomial logit (MNL) models, the random components are assumed to follow a normal, logistic and extreme value (Type I) distribution, respectively [The general form of the utility function is shown in equation (2.1).ectively .Current research into pedestrian route choice using utility theory aims to establish the relative utility of route attributes via measurements or experimentally, or it develops novel models based on the concept of utility. For example,  conducte. 2.4.2Despite their random component, utility models assume people hold knowledge of costs associated with all alternatives and perform an optimization across them. It has been suggested that this may not be an appropriate representation of the cognitive processes people perform ,67 calleA heuristic is a decision rule that does not seek to optimize and may ignore part of the information with the goal of effort reduction. In other words, heuristics allow people to make decisions quicker and with less cognitive effort using simple rules and inferences. It has been suggested that this reflects how individuals use cognitive shortcuts to reach intuitively correct decisions .A regular criticism of heuristics is that people save time and effort with a heuristic at the cost of accuracy. However, in some cases simple heuristics are more accurate than standard statistical methods that have the same or more information. When less information or computation leads to more accurate judgements than more information or computation, these results are known as less-is-more effects .Research has identified several heuristics pedestrians may use to make route choices  decline with age . They teThe contextual factors listed above illustrate the diversity of contexts and their contribution to shaping pedestrian route choice behaviours. Pedestrian route choice involves the pedestrian and the context itself as well as the interactions between them. Although the principles we identified can capture the key mechanisms and cross-domain relationships in this process, the role of these scenario factors should be considered when we apply these principles to a certain situation.. 3 (1)Pedestrians can perceive information selectively and purposely, given limited available information. (2)Pedestrians integrate environmental spatial information into mental representations with subjectivity. (3)Pedestrians tend to be attracted and repelled by specific attributes individually and this can lead to positive or negative feedback loops across many individuals. (4)Pedestrians perform trade-offs based on the evidence provided by different attributes. (5)How pedestrians perceive, integrate, respond to and decide upon information is not fixed but varies with the context.We identify principles that capture the essence of pedestrian route choice and are relevant across disciplines, and we give examples to demonstrate how these principles shape pedestrian choice behaviour. These principles are reiterated below:These proposed principles are reflected in models that researchers have developed. For example, the knowledge-based routing model in  construcMuch research into pedestrian route choice focusses on specific settings and does not consider aspects that are not immediately relevant to the research question investigated. While this reductionist approach forms an important part of the knowledge generation process and may be entirely appropriate to describe behaviour in fixed settings, such as carefully controlled experiments where participants are exposed to different stimuli in the same scenario, it is important to acknowledge the limitations of this approach. For example, a utility model can have an excellent performance in pedestrian route prediction even if selective information perception or/and feedback, the first principle we discussed, is not included in the model. However, such a model may not be useful for scenarios where pedestrians can only perceive limited information. We do not suggest that the principles we identify here provide a route to establish a universal model for pedestrian route choice. Instead, we suggest that our contribution provides a framework for considering which aspects of pedestrian route choice are accounted for in a given model.From the perspective of managing pedestrian facilities during events or emergencies, it is desirable to be able to predict or even control the route choice of pedestrians to minimize uncertainty and to maximize evacuation efficiency. Our principles provide a theoretical basis for developing route choice control or route guidance strategies. For example, taking the first principle as a starting point would suggest to selectively highlight information about particular routes. Alternatively, based on the third principle, routes could be made more attractive by changing their attributes, such as lighting or signage, or, based on the fifth principle, pedestrians could be made familiar with routes in a targeted way, as already happens on passenger airplanes. While many of these approaches are already being used or considered, we suggest that our principles provide a framework to structure and contrast strategies.More generally, we suggest the usefulness of our principles is that they provide a frame of reference that can be used to catalogue, contrast and analyse existing and planned research on pedestrian route choice. The broad behavioural mechanisms we identify facilitate abstracting from the details of behaviours observed and contexts investigated in individual studies. As such, they provide an ideal basis for categorizing and comparing existing work and for researchers to examine to what extent their planned work is already covered in the existing literature. For example, a large body of research focuses on how pedestrians respond to specific types of information during evacuation, and if researchers categorize their work into the third principle of our framework, they can find other similar research to avoid duplication of work and find potential reasons to interpret differences in results by comparing to other work. Alternatively, as discussed above, in transportation research there are many studies that investigate the attractiveness of route characteristics in experiments or from observations, but much fewer studies investigate the second principle, subjective mental representations of environments, which possibly influences human route choice and is worth further investigation. Our principles also provide a structured theoretical starting point for interpreting or explaining observed behaviours. For example, if it is observed that pedestrians completely avoid an available route, we suggest it is useful to structure the search for an explanation by considering our principles in turn. For example, it is possible that, based on the first principle, participants selectively ignore the information provided by the researchers, or based on the fourth principle, they choose their route according to specific heuristics, disregarding other options. It may be that future research identifies principles in addition to the ones we discuss here. This would be a clear indication of how our understanding of pedestrian route choice extends.Technological and methodological innovations will continue to facilitate research into pedestrian route choice. We suggest that three technologies in particular will shape this field of research in the coming years: virtual reality, wearable sensors and machine learning. Separately, and in combination these tools will allow examining the principles of pedestrian route choice in unprecedented breadth and detail. In virtual reality experiments participants interact with a highly controlled immersive virtual environment . The facPedestrian route choice is a highly interdisciplinary topic. Researchers from different disciplines are contributing to it by applying the methodologies and theoretical frameworks of their discipline. We argue our principles provide a general theoretical framework that facilitates bridging across disciplines.


The programmed −1ribosomal frameshifting element (PFSE)of SARS-CoV-2 is a well conserved structured RNA found in all coronaviruses’genomes. By adopting a pseudoknot structure in the presence of theribosome, the PFSE promotes a ribosomal frameshifting event near thestop codon of the first open reading frame Orf1a during translationof the polyprotein pp1a. Frameshifting results in continuation ofpp1a via a new open reading frame, Orf1b, that produces the longerpp1ab polyprotein. Polyproteins pp1a and pp1ab produce nonstructuralproteins NSPs 1–10 and NSPs 1–16, respectively, whichcontribute vital functions during the viral life cycle and must bepresent in the proper stoichiometry. Both drugs and sequence alterationsthat affect the stability of the −1 programmed ribosomal frameshiftingelement disrupt the stoichiometry of the NSPs produced, which compromiseviral replication. For this reason, the −1 programmed frameshiftingelement is considered a promising drug target. Using chaperone assistedRNA crystallography, we successfully crystallized and solved the three-dimensionalstructure of the PFSE. We observe a three-stem H-type pseudoknot structurewith the three stems stacked in a vertical orientation stabilizedby two triple base pairs at the stem 1/stem 2 and stem 1/stem 3 junctions.This structure provides a new conformation of PFSE distinct from thebent conformations inferred from midresolution cryo-EM models andprovides a high-resolution framework for mechanistic investigationsand structure-based drug design. Existing drugscan be screened against experimentally determined structural modelsusing structural dynamics simulations to identify potential binders.3The historic and deadlyCOVID-19 pandemic is caused by the SevereAcute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2). Researchersaround the world are searching for treatments for this catastrophicdisease, in part by targeting drug design efforts toward the structuredRNA elements in the nearly 30 kb RNA genome of the virus. However,a lack of high-resolution, three-dimensional structural informationabout structured regions of the genome make development of drugs totarget them difficult. Computational modeling and structural probingtechniques are able to identify structured regions within RNAs andcan suggest whether an RNA element might contain a pocket sufficientfor ligand binding, but these estimations often lack certainty aboutthe chemical arrangement of binding pockets.8 Chemical probing and homology modeling consistently predict thisprogrammed frameshifting element to form a stable and well-orderedthree-stemmed H-type pseudoknot.14 For structural biologists, its small size also makes it a promisingcandidate for X-ray crystallography. Thus, we saw an opportunity tosupport the drug design process and mechanistic investigations bygenerating a high-resolution structure of the programmed frameshiftingelement using our lab’s method of chaperone-assisted RNA crystallography.One potentially druggable RNA target inthe SARS-CoV-2 genome isthe programmed −1 ribosomal frameshifting element (PFSE). Thusfar, it has been shown to bind the ligand 2-{diazepane-1-carbonyl]-amino}-benzoicacid ethyl ester (MTDB), and in cell culture this ligand can compromiseribosomal frameshifting and inhibit viral replication by three ordersof magnitude.18 Orf1a and its out-of-framecontinuation Orf1b are the first open reading frames to be translateddirectly from the SARS-CoV-2 RNA genome upon infection and disassemblywhich corresponds to an A in position 13533 in SARS-CoV-2.59 The pseudoknot structure of theframeshifting element from SARS-CoV-1 has been well-characterizedpreviously by chemical probing, mutational, and NMR studies of minimalconstructs.21 The secondary structure of the SARS-CoV-1 PFSE derived from thesestudies that includes a hepta-nucleotide slippery site, nucleotides 13462–13468in the genomic numbering, and a spacer region followed by the proposedRNA pseudoknot structure in positions 13474–13542 in the genome1A.30 Intstudies 1Bhas be35 Small-angle X-ray scattering diffraction analysisby Kelly et al. has shown that the SARS-CoV-1 frameshifting pseudoknotand the SARS-CoV-2 frameshifting pseudoknot have nearly identicaltopology.33 Functionally, both SARS-CoV-1and SARS-CoV-2 pseudoknots frameshift to appreciable rates in vitro and in cells.33 Likewise,in both cases, frameshifting is inhibited in the presence of the ligandMTDB, which implies that both PFSEs bind the ligand.33 This evidence allows us to apply the established research on theSARS-CoV-1 PFSE to this new SARS-CoV-2 PFSE in our investigation ofthe structure.Despite the one nucleotide substitution, the SARS-CoV-2 PFSElikelyadopts the same conformation as the SARS-CoV-1 PFSE. Computationalmodeling predicts similar structures for both sequences, and NMR studiesshow close agreement with the predicted three stem structure in bothcases.To contribute higher resolution information aboutthe SARS-CoV-2PFSE structure, we have applied the chaperone assisted RNA crystallographymethod to the PFSE. We created a modified construct of the PFSE lackingthe upstream slippery site and spacer region. This structured RNAwas then bound to an engineered humanized murine antibody fragmentto facilitate cocrystallization. Using iridium hexammine, we solvedthe structure using a combination of single angle anomalous diffractionphasing and molecular replacement. Here, we report the structure ofthe SARS-CoV-2 programmed frameshifting element pseudoknot solvedby X-ray diffraction to 2.09 Å.30 Our crystallization constructincludes only the minimal sequence for the pseudoknot (13474–13541).We omitted the spacer and the slippery site sequences from the crystallizationconstruct due to the high flexibility expected for single-strandedregions at the ends of RNAs, which can reduce crystallization efficiency.In this construct, the starting residue, nucleotide 13474, was mutatedfrom U to G to enhance in vitro transcription efficiency.This residue is predicted to be unpaired and is not considered essentialfor pseudoknot formation. It was unclear whether the final uridineof the pseudoknot sequence (residue 13542) would be paired.9 To avoid a dangling nucleotide in the construct,which can compromise the crystallization construct rigidity, thisnucleotide was excluded from the template sequence. However, uponsequencing RNA extracted directly from the crystal, we found thatthe construct that crystallized had an untemplated G added to the3′ end . We refer to this minimal version of the PFSE as the wildtype PFSE construct.The PFSE regioncomprises the so-called slippery site (13462–13468),followed by a spacer region and finally the core pseudoknot spanningnucleotides 13474 to 13542 in the genomic numbering for strain HB011A.12,15,37 Among these, Fab BL3–6binds to hairpins with the loop sequence (AAACA) closed by a GC pair,which we call the BL3–6-binding epitope. The most suitablelocation to graft the BL3–6-binding epitope into the PFSE wasloop 2, at the base of stem 3. The SARS-CoV-1 PFSE has been shownto retain wild type levels of frameshifting activity in vitro when loop 2 was replaced with a GUUG tetraloop; therefore we expectedthat the PFSE structure could tolerate an AAACA pentaloop in the loop2 position.14 We refer to this Fab-bindingPFSE construct as PFSE BL3–6. In an electrophoretic mobilityshift assay (EMSA), the PFSE BL3–6 construct formed a mobilityshifted species in the presence of Fab BL3–6, suggesting thatthe grafted sequence formed the expected loop and had not alteredthe predicted RNA secondary structure as shown in Supporting Informaiton Figure 4 .RNA elements often resist forming well-diffractingcrystals due to their biophysical properties such as instability,negatively charged backbone, conformational heterogeneity, and limitedfunctional group diversity for mediating specific lattice contacts.Our lab has found that antibody fragment (Fab) RNA complexes crystallizemore readily than RNA alone. In previous work, we reported developinga suite of humanized murine antibody fragments and their cognate RNAmotifs, which can be grafted into structured RNA targets to createa Fab binding site.–1 and crystallized in 2% v/v tacsimate at pH 4, 0.1M sodium acetate trihydrate at pH 4.6, and 16% w/v polyethylene glycol3,350  and was further optimizedwith the addition of either 0.01 M sarcosine or 0.01 M betaine hydrochloride.Some crystals were soaked in cryogenic protectant containing iridiumhexammine to aid in phasing using anomalous dispersion. These crystals,with and without heavy metal soaking, yielded multiple data sets withthe best diffracting to 2.09 Å. To solve the structure, datasets with a strong anomalous signal and low resolution were phasedin Phenix using a combination of single-wavelength anomalous diffraction(SAD) experimental phasing and molecular replacement using the FabBL3–6 model.40 An initialelectron density map was generated, and a partial model of the RNAwas then built in COOT.41 This partialRNA model and the Fab model were then used to find a molecular replacementsolution for a separate high resolution native diffraction data set.This solution gave complete electron density maps. Iterative roundsof building and refinement in this high-resolution data set yieldeda complete structure for the PFSE pseudoknot at 2.09 Å with an Rwork of 19.93% and an Rfree of 23.08% , a feature inferred from midresolutioncryo-EM models  of stem 2 to form the long-distance H-type pseudoknotinteraction. Starting from the 5′ end of loop 1, nucleotideU13485, which was predicted to be unpaired 2A, formsG13486 engages in a base triple interaction with G13493 andA13537.G13486 uses its Hoogsteen face to interact with the Watson–Crickface of G13493 and uses its exocyclic keto group to accept a hydrogenbond from A13537’s 2′ hydroxyl group 3F. We coStem 2 was originallypredicted to consist of six base pairs between nucleotides 13488–13494and nucleotides 13542–13535 with A13537 bulged out from thehelix 3A.Howevin vitro transcribed RNAs extracted from crystals to confirm the presenceof an additional nucleotide and reveal its identity.42 Briefly, the RNA was polyadenylated to create a primerbinding site for a poly(T) reverse primer for reverse transcriptioninto cDNA. The resulting cDNA was amplified and sequenced. This notonly revealed the identity of the terminal residues but also quantitativelymeasured the enrichment for each of the four nucleotides randomlyadded by T7. RNA extracted from washed crystals shows a G predominatingafter C13541 . By contrast themother liquor contains transcripts with a mix of G and C . The enrichment for G over C in thecrystal may be due to the crystal contact involving stacking of G13542with a mirrored symmetry mate of the RNA. Whether the native U inposition 13542 forms a base pair with A13488 remains unclear.9Although the DNA template used fortranscription of the PFSE construct was designed to terminate transcriptionat nucleotide 13541, the electron density map showed density for anadditional 65th nucleotide. As T7 RNA polymerase is known to add untemplatednucleotides to the 3′ terminus of transcripts, we used a simplemethod to sequence the 3′ ends of 35 This residue is often modeled as unpaired in Loop 3 or paired toU13504 in Stem 3. However, in this structure we see clear densityfor a triple base pair involving G13475, U13504 and A13533 at theS1/S3 junction . Thus, the SARS-CoV-1 PFSE also appearscapable of forming a triple base interaction at the S1/S3 junction,supporting the functional relevance of the base triple.Compared to the PFSEof SARS-CoV-1, the PFSE of SARS-CoV-2 hasa single C to A substitution at residue 13533 in SARS-CoV-2 whichcorresponds to 13444 in SARS-CoV-1. The C to A mutation itself seemsto have no effect on frameshifting efficiency as both frameshift toappreciable extents, although the in vitro and in vivo efficienciesvary significantly.43 Ourstructure shows that threading is due to the twist of the stem 1 helixholding the first five nucleotides in place as loop 3 and the 3′end of the RNA fold over top . Here, we also find that G13475, included at the 5′ endfor transcriptional efficiency in place of the native U, interactswith the G13505–U13532 wobble pair of stem 3 . In this base triple, the N1H imino group and N2H2 exocyclic amine of G13474 donate hydrogen bonds to N7 andthe O6 keto group of G13505, respectively . The native U nucleotide in position 13474 would not beable to make the analogous interactions. While the non-native interactionof G13474 may stabilize the vertical conformation, its absence inthe native structure would not preclude formation of the verticalconformation.TheS1/S3 junction is also the site of the 5′ end threadingthough the ring created by stem 1 and loop 3, consistent with observationsfrom other structural studies of the PFSE.44 Single-strandedRNA is expected to be more compact than ssDNA due to steric restraintsimposed by the 2′ OH on the sugar pucker conformations. Ifnucleotides 13534, 13535, and 13536 in loop 3 were fully extended,they would span a maximum distance of 20.1 Å, leaving only ∼2.7Å of slack for bending. In a study of a 40-nucleotide strandof poly(U) RNA, a total contour length of 196 Å was measured,giving 4.9 Å as the average length of each nt.44 In a relaxed state, we would therefore expect three nucleotidesto span 14.3 Å, meaning that in spanning 17 Å the nucleotidesin loop 3 are likely extended and less flexible than they would beif their ends were free. We anticipate that the residues in this regionlikely possess enough flexibility to wiggle back and forth like ashort string held at both ends but must maintain a relatively extendedbackbone conformation to bridge the distance between residues 13533and 13537.Loop 3, which connects stems 2 and 3,consists of nucleotides 13533, 13534, 13535, 13536, and 13537 in ourstructure. Density for residues 13534, 13535, and 13536 is missingfrom the data sets, suggesting this loop is flexible and not wellorganized in the crystal 3D. Never14 We find this residue unpaired and stacked withinstem 3 .4343 Our 2.09Å structure confirms many of the base pairing interactions thatremained ambiguous at 6.7 Å. The secondary structures reportedby each work identify the same differences from the “literature”secondary structure as we have found. The structures agree that stem2 ends at A13537, and the crystal structure reveals additional detailsabout the specific base pairing interactions at the S1/S2 helicaljunction. Our structure also shows the same 5′ end threadedtopology first identified in these cryo-EM structures. In contrastto the cryo-EM structures, our crystal structure adopts a verticallystacked conformation, which has been consistently predicted by computationalmodeling but not yet observed experimentally.34 This structure could represent the secondSARS-CoV-2 PFSE conformation.Both cryo-EM structures find the PFSE ina “bent” arrangement, where stem 1 and stem 2 are helicallystacked while stem 3 bends perpendicularly away from stem 1.43 Additionally,in the ribosome-bound structure, G13486 forms direct interactionswith the N-terminal domain of US3 while A13537 remains unpaired inthe J2/3 region.4 Therefore, the S1/S2base triple we observe would have to be dissolved in the bent conformationand in the ribosome bound state. Interestingly, mutations to G13486or A13537 in isolation were shown to reduce frameshifting frequencymarkedly, although the role of A13537 is unknown.4 The PFSE’s intolerance to A13537 mutations combinedwith these structural observations suggest that G13486 may serve anadditional structural role to orient G13493’s sugar edge tointeract with A13533’s WC face. In other −1 frameshiftingpseudoknots, elimination of known base triple interactions does reduceframeshifting efficiency by destabilizing the pseudoknot.48 These observations suggest that the S1/S2 basetriple may act as a conformational switch or decision point betweenthe bent conformation and the vertical conformation.Curiously, neither of the basetriple pairing interactions we reportare present in the cryo-EM models, although this may be due to thelimited resolution of these structures. The bent conformation of thePFSE and slippery site observed in the solution cryo-EM model (PDBcode: 6XRZ)positions the S1/S2 base triple nucleotides too far apart to form the base triple 5C.43 Add4 In the cryo-EM structure ofthe element in isolation (PDB code: 6XRZ), G13475 and U13504 are close enoughto base pair, while A13533 is more than 10 Å away  in SARS-CoV-1. The role of this C to A substitutionis not yet understood, but when A13533 is modeled as a C, the hydrogenbonding interactions with G13475 can still be formed .33 Therole of this S1/S3 junction region now warrants deeper investigationas it could dictate a dynamical relationship between bent and linearconformations and thereby influence frameshifting efficiency.The basetriple involving G13475, U13504, and A13542 at the S1/S3junction may further stabilize the vertical conformation as it cannotbe fully formed in the bent or ribosome-bound cryo-EM structures.In the ribosome bound structure, G13475, of the S1/S3 base triple,is inside the mRNA entry channel, but A13533 and U13504 are modeledas paired.0 Å away 5D.43 Unf10 Indeed, substantial data exist that support the hypothesis thatthe frameshifting element populates two distinct conformations thatgovern the efficiency of PFSE frameshifting.50Our crystal structure, the cryo-EM structure of the free PFSE withthe slippery site, and the ribosome-bound PFSE-containing gRNA fragmenthave similar topologies and differ only at junctions where alternatepairing interactions or ribosomal interactions can occur. The crystalstructure model represents a vertically stacked conformation, andthe cryo-EM structures represent a bent or wedged conformation. Modelingof the PFSE by Omar et al. and Rangan et al. demonstrated that thearrangement of stem 1 and stem 2 relative to stem 3 can be flexible.58 Rather than frameshifting itself, the vertical conformationobserved here could have significance for PFSE structural rearrangementsthat occur during other stages of the virial lifecycle. Moreover,as there are limited structures available for RNA pseudoknots, thehigh-resolution structure data presented herein have intrinsic valuefor the structure-based design of small molecule binders, as a startingpoint for molecular dynamics simulations, and as a framework for testingthe relationship of the RNA structure to its function.We caution, however, that conformational differencesdetected acrossthe various models of the PFSE may be due to differences in RNA constructdesign and or ribosomal interactions. For example, our construct lacksthe 5′ slippery site sequence, which forms a helix that coaxiallystacks beneath stem 1 in the cryo-EM model. The slippery site helixcould preclude formation of the vertically stacked conformation justas the ribosome does. Additionally, recent investigation of the SARS-CoV-2RNA genome both within the cytoplasm and the virion infers entirelynew secondary structures for the PFSE.49 Frameshiftingpseudoknots must also be conformationally dynamic to function, refoldinginto one among multiple conformations each time the ribosome readsthrough the mRNA.49 Optical tweezers can be usedto both unfold the pseudoknot and mimic the tugging forces the ribosomewould apply to a pseudoknot during translation.29 In reporter assays, the rate of frameshifting of any given pseudoknotcorrelates linearly with its conformational Shannon entropy, a statisticalmetric for conformational plasticity.29 Pseudoknots that occupy two conformations often induce frameshiftingon the order of 20% efficiency.27 Nevertheless,frameshifting rates measured in vitro can be differentthan those measured in vivo or in infected cellsdue to additional interactions with proteins and the gRNA.In programmed ribosomal −1 frameshifting pseudoknots,helical stacking is often a required feature to induce frameshifting,and tertiary interactions have been shown to stabilize frameshiftingpseudoknots and promote efficiency.50 The more stable form (N) unfoldsat an average force of 30 pN, and the slightly less stable form (N′)unfolds at an average force of 16 pN. Possibly, the two states correspondin part to the vertical and bent conformations, although we emphasizethat this awaits careful validation. The base triples in the verticalconformation would be expected to provide additional stability andrigidity relative to a conformer lacking them. In line with this possibility,another H-type pseudoknot found in human telomerase RNA (ΔU177)was found to lose ∼16 pN of unfolding force upon disruptionof two minor groove base triples, which is a similar difference inunfolding force between N and N′.50 It will be important to address whether the vertical conformationcorresponds to one of the states in the unfolding experiments, andour structure will inform the design of atomic mutations for thesetests.Unfoldingforce extension curves show that the SARS-CoV-2 PFSE can adopt twodistinct conformations of roughly similar stability, falling in linewith expectations given a frameshifting frequency in the 15–30%range.30 We chose to exclude nucleotide 13542 in thiscrystallization construct because its binding to A13488 was unclear,and incorporating an unpaired nucleotide at the 5′ end couldcompromise the structural integrity of the crystallization complex.Typically, the PFSE is defined as this region as well as the 14 nucleotidesupstream, which include the slippery site; these were excluded fromthe crystallization construct because long single-stranded regionsof RNA can disrupt folding and crystallization of the RNA. This truncatedconstruct we refer to as the wild type PFSE construct.Our crystallization target was basedon nucleotides 13475–13541 in the SARS-CoV-2 genome, Gen Banknumber NC_045512.2; these are the minimal residues predicted to form base pairs.To enablebinding to antibody fragment, Fab BL3–6, we mutated the nucleotidecorresponding to loop 2 (13514 to 13522) to the sequence AAACA. Thiscrystallization construct is referred to as the BL3–6 PFSEconstruct. We chose to mutate these residues because mutational studieshave shown that loop 2 can be mutated to a common RNA tetraloop withoutaltering the ratio of frameshifting in the SARS-CoV-1 PFSE, whichis believed to be structurally identical to the SARS-CoV-2 PFSE.51 predictions suggest that theBL3–6 PFSE does not contain non-native regions of complementaritythat might be prone to disrupt the native secondary structure. A gelshift assay was performed on refolded PFSE RNA constructs and antibodyfragment BL3–6 to show that the grafted nucleotides still formthe expected solvent-exposed loop, and that Fab binding was only observedfor the mutated construct.V-fold52 Transcription templateDNA was amplified into double-stranded DNA using PCR. RNA was transcribedfrom the purified PCR product using an in vitro transcriptionreaction; 50 pmol mL–1 DNA template was incubatedfor 3 h at 37 °C in buffer containing 40 mM Tris-HCl, at pH 8.0,2 mM spermidine, 10 mM NaCl, 25 mM MgCl2, 10 mM DTT, 40U mL–1 RNase inhibitor, 5 U mL–1 thermostable inorganic pyrophosphatase, 5 mM of each NTP, and 50μg mL–1 T7 RNA polymerase. Reactions werehalted by the addition of RNase free DNAase1 at 5U mL–1 and incubation at 37 °C for 30 min. RNA was purified on a 10%denaturing polyacrylamide gel in 0.5 × TBE running buffer. TheRNA was visualized with UV shadowing, extracted, and eluted into 10mM Tris, at pH 8.0, 2 mM EDTA, and 300 mM NaCl buffer via overnightincubation at 4 °C. The eluted RNA was then concentrated andexchanged into double distilled H2O using a 10K Amiconfilter and stored at −80 °C until further use.Single strandedDNA templates and primers for PCR and transcription were ordered fromIDT encoding the transcription template for each RNA construct witha T7 promoter. Forward primers were ordered matching the T7 promoterregion, and reverse primers contained a single 2′ O-methylmodification at the 3′ end to avoid untemplated additions byT7 polymerase.www.atcc.org) and grownon LB plates supplemented with carbenicillin at 100 μg mL–1. Nine colonies from the plates were chosen and inoculatedto a starter culture with 100 μg mL–1 carbenicillin,which was grown at 30 °C for 8 h. Once the starter culture reachedan OD 600 of 8, 15 mL of starter culture was used to inoculate 1 Lof 2× YT media and grown for 24 h at 30 °C. The cells werethen pelleted via centrifugation at RT, and the cell pellet was resuspendedin 1 L of freshly prepared phosphate depleted media supplementedwith 100 μg mL–1 carbenicillin. The cellswere set to grow for 24 h at 30 °C, harvested via centrifugationat 4 °C, and frozen at −20 °C. Frozen cell pelletswere lysed in PBS buffer supplemented with 0.4 mg mL–1 of lysozyme and 0.01 mg mL–1 of DNase I. After30 min, PMSF was added to a final concentration of 0.5 mM. After 30min, the cells were centrifuged for 45 min at 12 000 rpm and4 °C. Lysate was transferred to new sterile bottles and centrifugedagain for 15 min at 12 000 rpm and 4 °C. Supernatant wasfiltered through 0.45 μm filters into a sterile bottle , and Fab proteins were purified using the AKTAxpress fast proteinliquid chromatography (FPLC) purification system as described previously.37 The lysate inPBS buffer (pH 7.4) was loaded into a protein A column, and the elutedFab in 1 M acetic acid was buffer exchanged back into the buffer PBS(pH 7.4) using a 30 kDa cutoff Amicon filter and loaded into a proteinG column. The Fab was eluted from a protein G column in 0.1 M glycine(pH 2.7) and then buffer-exchanged into 50 mM NaOAc and 50 mM NaClbuffer (pH 5.5) and loaded into a heparin column. Finally, the elutedFab in 50 mM NaOAc and 2 M NaCl (pH 5.5) was dialyzed back into 1×PBS (pH 7.4), concentrated, and analyzed with 12% SDS-PAGE using CoomassieBlue R-250 staining for visualization. Aliquots of Fab samples weretested for RNase activity using the RNaseAlert kit . Thealiquots of Fab samples were flash frozen in liquid nitrogen and storedat −80 °C until further use.The BL3–6 Fab expression vector(available upon request) was transformed into 55244 chemically competentcells  was added,and the RNA was then incubated at 50 °C for 10 min to facilitaterefolding. RNA was then mixed with either PBS as a negative controlor a 1.1 M ratio of RNase–free Fab BL3–6 and incubatedat RT for 30 min to establish equilibrium binding. Fab RNA complexeswere separated by gel electrophoresis in a 12% polyacrylamide gelmade in 0.5× TBE buffer supplemented with 5 mM MgCl2. The gel was stained with ethidium bromide and visualized via UVlight and photographed .To validateFab binding to the BL3–6 PFSE RNA construct, purified RNA constructsin double distilled HE. coli poly(A)polymerase. For the mother-liquor-derived RNA samples, mother liquorwas harvested from a 200 nL drop of crystallized complex. To ensurethat all of the mother liquor solution was harvested, 0.5 μLof well solution was added to the drop prior to transferring the samplesto an Eppendorf tube. For the crystal-derived RNA samples, after themother liquor was harvested from the drop, the remaining crystalswere washed three times with well solution. Then, the crystals weretransferred to an eppendorf tube with 2 μL of RNase free waterand crushed via pipetting. Samples with transcribed RNA and motherliquor- and crystal-derived RNA were denatured for 1 min in 90 °C.Then, they were cooled down on ice for 2 min and incubated for 3 minat RT. Denatured RNA samples were elongated with 1 μL of E. coli poly(A) polymerase 5000 U mL–1 (NEB), and 2 μL of 10× E. coli poly(A)polymerase reaction buffer with the addition of 2 μL of 10 mMATP and RNase free water up to 20 μL as per the manufacturer’sinstructions. The reaction was incubated at 37 °C for 30 minin a water bath. The reaction was halted by the addition of EDTA toa final concentration of 10 mM. RNA was ethanol precipitated and checkedfor poly(A) elongation on a 10% polyacrylamide gel stained with ethidiumbromide. Polyadenylated RNA was used as the template in a reversetranscription reaction using SuperScript III (Invitrogen) accordingto the manufacturer’s instructions. cDNA was amplified usingend specific primers and PCR with 30 cycles of amplification usingan annealing temperature of 52 °C and Taq DNApolymerase (NEB). The double stranded DNA products were then submittedfor sequencing. We note that this method is one of many commonly usedto sequence the 3′ end of RNAs.57To determine the identity of theuntemplated nucleotide observed in the electron density map, we sequencedthe RNA from three sources: the transcription product, the motherliquor of the crystal drop, and the crystal itself. For the transcribedRNA, we followed the standard procedure for poly(A)-tailing (NEB)using 3 μg of RNA in reaction with 2, 100mM KCl) and incubated at 50 °C for 10 min. Fab was added to theRNA at a 1:1.1 molar ratio of RNA/Fab and incubated at RT for 30 minbefore concentrating the complex to 6 mg mL–1 RNAvia centrifugal filtration in a 10 kDa cutoff Amicon Centrifugal FilterUnit. Concentrated complexes were then filtered using 0.2 μmcutoff Millipore centrifugal filter units and used to set high-throughputhanging-drop vapor-diffusion crystallization screens at RT using commerciallyavailable screening kits from Hampton Research and Jena Bioscienceusing the Mosquito liquid handling robot (TTP Labtech). Crystals grewin 2% v/v tacsimate at pH 4. 0.1 M sodium acetate trihydrate at pH4.6, and 16% w/v polyethylene glycol 3,350 and were further optimizedwith the addition of either 0.01 M sarcosine or 0.01 M betaine hydrochloride.Crystals appeared and grew to full size within a week at 21 °C.PFSE BL3–6 RNA was denaturedin water by incubation at 90 °C for 1 min, in ice for 2 min,and at RT for 3 min. The RNA was then refolded by the addition ofrefolding buffer  and 3 mM iridium hexammine as a cryo-protectantand to incorporate iridium hexammine into the crystal lattice. Iridiumhexammine was synthesized in-house following the protocol establishedby Batey et al.https://rapd.nec.aps.anl.gov). Initial SAD-MR phases were obtained from a data set which diffractedto only 3.27 Å using a partial molecular replacement (MR) solutionof the Fab (PDB code: 6DB8) in Phenix Autosol.40 A low-resolutionelection density map was able to be calculated for a portion of theRNA from which a partial model of the FSE was built. This partialRNA model was used in addition to the fab model (PDB code: 6DB8) to find a molecularreplacement solution for a higher resolution (2.09 Å) data setcollected from a native unsoaked crystal. The electron density mapusing the MR phases of the partial RNA-Fab model were vastly improvedand allowed for unambiguous model building nucleotide by nucleotide.38 The model was iteratively built and refined in COOT and Phenix Refineuntil the Rwork and Rfree could not be further improved.41 Water was automaticallyadded during refinement and later validated visually in COOT41 according to the electron density map and differencemap and potential hydrogen bonding interactions. All structure relatedfigures were made in PyMOL54 (www.pymol.org), and figure labelswere edited in Microsoft PowerPoint.The X-ray diffractiondata sets were collected at the Advanced Photon Source NE-CAT sectionbeamline 24-ID-C and 24-ID-E. Crystals soaked with iridium hexamminewere shot with both the default wavelength of 0.979180 and a wavelengthof 1.04040 to illicit anomalous diffraction of the iridium. All ofthe data sets were then integrated and scaled using its on-site RAPDautomated programs (


RNA molecules are increasingly being identified as facilitating or impeding the interaction of proteins and nucleic acids, serving as so-called scaffolds or decoys. Long non-coding RNAs have been commonly implicated in such roles, particularly in the regulation of nuclear processes including chromosome topology, regulation of chromatin state and gene transcription, and assembly of nuclear biomolecular condensates such as paraspeckles. Recently, an increased awareness of cytoplasmic RNA scaffolds and decoys has begun to emerge, including the identification of non-coding regions of mRNAs that can also function in a scaffold-like manner to regulate interactions of nascently translated proteins. Collectively, cytoplasmic RNA scaffolds and decoys are now implicated in processes such as mRNA translation, decay, protein localization, protein degradation and assembly of cytoplasmic biomolecular condensates such as P-bodies. Here, we review examples of RNA scaffolds and decoys in both the nucleus and cytoplasm, illustrating common themes, the suitability of RNA to such roles, and future challenges in identifying and better understanding RNA scaffolding and decoy functions. The cell, with its variety of cellular compartments, varying polarities, competing interactions, and differing sites of molecular synthesis poses challenges to the formation of biomolecular interactions essential to all biological processes. This necessitates ways to bring biomolecules together in a tightly regulated manner. One mechanism for this localization is the use of molecular scaffolds to enable particular interactions while inhibiting off-pathway interactions, thus increasing the efficiencies of the biological processes in which they are involved in In this review, we define an “RNA scaffold” as an RNA molecule capable of bringing together 2 or more macromolecules to form a complex with functional activity. These macromolecules may be proteins, other RNAs or DNA molecules that in the absence of the RNA scaffold do not interact or do so very poorly. By this definition, perhaps the most well-known RNA scaffold molecules are ribosomal RNAs (rRNAs), which provide the structural and catalytic core of ribosomes around which approximately 57–78 ribosomal proteins (depending on species)  assembleRNA can also function as a “decoy” molecule, which we define similarly to a scaffold, except that in this case, two or more macromolecules are brought together by the RNA decoy in a complex that prevents the sequestered macromolecules from forming other interactions and functional complexes at other cellular locations. As with scaffolds, RNA decoy interactions can be transient and regulatable.RNAs as molecular scaffolds or decoys possess several advantages over other types of biomolecules like proteins and DNA, including: (1) RNAs fold more readily than DNA into complex 3D structures by virtue of diverse secondary and tertiary structural interactions. (2) RNA abundance can be easily and rapidly increased (new transcription) or decreased (RNA decay) while using 1–2 orders of magnitude less energy than occurs for similar regulation of protein ; indeed In this review, we discuss examples of many nuclear and cytoplasmic RNA scaffolds and decoys, some of which regulate relatively stable complexes, while others facilitate transient macromolecular interactions. Concepts of RNA-driven assemblies that have emerged, their physiological importance, and key remaining questions for future study will be highlighted. We will also focus on recent findings of mRNAs acting as cytoplasmic RNA scaffolds, and other means by which nascently translated proteins find their interaction partners. Finally, we discuss steps that can be taken to identify new RNA scaffolds and decoys.RNA scaffolds can generally be classified as either “structured” or “flexible.” RNAs in the former class tend to scaffold stable complexes and are enriched in secondary/tertiary RNA structure, while RNAs in the latter class often aid more transient macromolecular interactions and tend to be less structured.In this category, the RNP, composed of the RNA scaffold along with its partner proteins, is sufficiently stable in structure that it can be assessed by X-ray crystallography or by high resolution cryo-electron microscopy (cryo-EM). Zappulla and Cech  have sug(i)Structure determined mostly by RNA: In this category, the RNP structure is determined in large part by the folded RNA. An example of this is the ribosome, in which disordered tails or internal loops of proteins constituting the RNP are ordered only after they bind to folded rRNA . The rRN(ii)Structure determined mostly by protein: The RNP has a specific structure that is determined in large part by previously established protein-proteins interactions that constitute the RNP, evident in small nuclear RNPs (snRNPs) and small nucleolar RNPs (snoRNPs). Several snRNP and snoRNP proteins can be crystallized in heteromeric complexes in the absence of their RNA component . That saThe rest of this review will focus on flexible RNA scaffolds, which nucleate RNPs that lack a rigid structure. In this case, the primary role of the RNA is typically to maintain spatial proximity of macromolecular components, and aid interactions that are often  more transient in nature. Such RNAs often lack strong sequence conservation and can tolerate large deletions or insertions, but remain functional . ConversMost known RNA scaffolds and decoys are defined as lncRNAs, which are a heterogenous group of RNAs > 200 nucleotides in length, and which generally lack long conserved open reading frames, though some may encode short peptides . lncRNAsAn elegant example of an RNP featuring a flexible RNA scaffold is the telomerase enzyme that functions in the maintenance of genome stability by counteracting the loss of DNA at chromosomal ends following rounds of DNA replication . TelomerTelomerase complex assembly, and particularly recruitment to telomeres is often transient, and typically regulated in a cell-cycle dependent manner . In yeasMany lncRNAs associate with chromatin, and can enact either widespread or local changes in chromatin conformation that can regulate chromosome topology, localization and the process of transcription .Xist (X-inactive specific transcript), a 17 kb lncRNA that mediates X chromosome inactivation (XCI), a method of sex-chromosome gene dosage compensation where one X chromosome in every female cell is almost completely transcriptionally silenced at random (Xist lncRNA coats and is tethered to the entire future inactivated X chromosome (“Xi”) by an AT-rich DNA interacting protein called hnRNP U , a trans-membrane protein embedded in the inner nuclear membrane    and can tor CTCF . Firre ator CTCF .trans-chromosomal interacting loci , in cis or in trans, by base pairing with other RNAs, by the lncRNA binding directly to DNA, or by RNA-protein interactions  was identified as an Xist binder which in turn recruits SMRT (silencing mediator of retinoid and thyroid receptor) and histone deacetylase 3 (HDAC3) , which is bound by the RNA-binding protein (RBP) hnRNP K  . More re hnRNP K . This pr hnRNP K , a featu hnRNP K . Interes hnRNP K , though in trans at specific gene loci; this was first reported for the 2.2 kb human HOX antisense intergenic RNA (HOTAIR), which is encoded on chromosome 12 at the HOXC locus  HOTAIR XC locus .LncRNAs can also directly affect transcription by interacting with transcription factors and the transcriptional apparatus. For instance, in breast cancer cells, HOTAIR scaffolds a complex involving LSD1 , b and HlncRNAs can function as RNA scaffolds for nuclear bodies , which aNuclear paraspeckles, which are implicated in regulating transcription, splicing, RNA processing and export, are scaffolded by the nuclear-enriched abundant transcript 1 (NEAT1) lncRNA . ParaspeNEAT1 and paraspeckles regulate gene expression in diverse ways through both scaffolding and decoy functions. As a scaffolding example, NONO-SFPQ heterodimeric complexes bind to and enhance processing of select pri-miRNAs by the Microprocessor complex, which conducts the first step in miRNA processing . NEAT1 bA decoy function of paraspeckles and NEAT1 is sequestration, and thus limiting export of mRNAs harboring inverted repeat Alu elements in their 3′ untranslated region  experiments, which identified a strong enrichment of Alu repeat-containing RNAs (AluRNAs) in nucleoli that mostly derive from introns and subsequent pre-mRNA splicing of multiple genes  is a well-studied RNP complex that is scaffolded by an RNA Polymerase III-derived RNA 7SL; . SRP recThe human 7SL lncRNA is a ∼300 nt long structured RNA characterized by helical regions forming 2 domains separated by a flexible linker, a smaller domain (Alu) and a larger domain (S), with each part recruiting specific proteins  that form the SRP . The AluWhile most lncRNAs exhibit a significant bias toward nuclear localization, many also localize in the cytoplasm and thus unsurprisingly, are increasingly being linked to an array of diverse scaffolding and decoy roles.in trans, and scaffold complexes that impact mRNA function. For example, numerous lncRNAs harboring Alu elements (“1/2-sbsRNAs”) were identified that form partially complementary duplexes in trans with Alu elements present in various mRNA 3′UTRs  gene. BIRC3 encodes a ubiquitin ligase, and alternative cleavage and polyadenylation generates short (BIRC3-SU) and long (BIRC3-LU) 3′UTR mRNA isoforms, the latter of which is significantly upregulated in chronic lymphocytic leukemia (CLL) cells . While nWhile best known for its nuclear function HOTAIR is also found in the cytoplasm, particularly under cellular conditions such as senescence . CytoplaConversely, a well appreciated mode of lncRNA function in the cytoplasm is to function as decoys for miRNAs  interacts with nascently synthesized protein translated on the mRNA itself, with important physiological consequences . The firRPS28B, one of two RPS28 paralogs  that has an unusually long 3′UTR (643 nts) and which harbors a stem-loop structural element which binds Edc3  will possess potent scaffolding potential for RNP granules, based on their potentially high interaction valency  and abiolecules . RPS28B criteria , though e 3′UTRs  a broad e 3′UTRs .While mRNA localization clearly facilitates protein localization , co-locaIn human cells, several membrane protein-encoding mRNAs, including CD47-LU (but not CD47-SU), harbor binding sites for an RBP called TIS11B. These mRNAs, as well as SET and HuR, coalesce with TIS11B in an ER-intertwined reticular structure termed TIS granules, that rely on TIS11B expression for their assembly . ImportaIn yeast, specific glycolytic mRNAs  and seleEstimates in the human genome suggest between 20 and 60k loci in the human genome transcribe lncRNA , with ovAssessing reproducibility and condition-specific changes in expression levels, as well as conservation of RNA sequence, exon number, genomic position and transcriptional level is a generally utilized approach for identifying putative functional lncRNAs, for which various amenable bioinformatic tools are now available . LncRNAsKnown scaffold or decoy RNAs often harbor structured domains and typically interact with proteins. RNA-seq technologies that assess RNA secondary structure  has been used to identify or validate prediction of TR RNAs in several species . Cross-lFinally, microscopy-based analyses using FISH, in combination with immunohistochemistry of other cellular organelles or proteins, can provide insight as to functional and regulatory mechanisms of lncRNAs. A study of 61 lncRNAs in various human cell types revealed that while most were nuclear, highly diverse patterns of localization were observed . NotablyThe above methods can also be applied to identifying mRNA scaffold and decoy molecules, though separating 3′UTR-based regulation of nascent protein-interaction from 3′UTR based regulation of the mRNA  provides significant additional challenges. Thus, perturbation or forced expression of a mRNA 3′UTR isoform of interest, coupled to assessment of protein interactions  should include control experiments to verify no alteration of mRNA function or overall protein expression.It is clear there is no one single path to identification of functional RNA scaffolds and decoys. While sequencing, bioinformatic and comparative analyses studies generally offer the most rapid means to identify putative RNA scaffolds and decoys, genetic, biochemical and microscopy methods can all be brought to bear to enhance mechanistic understanding.The evidence is mounting that RNA molecules ubiquitously function as scaffolds and decoys bringing together or sequestering specific biomolecules to regulate macromolecular complex assembly. LncRNAs primarily accumulate in the nucleus , which hin trans) of RNA scaffolds and decoys is regulated in response to changing cellular environments is lacking in most cases. Additionally, while several RNA scaffolds and decoys, particularly within the category of lncRNAs, have been identified and functionally characterized in mammalian model systems, fewer characterized examples exist in other model systems. How, or indeed if, RNA-scaffolded complexes are actively assembled and disassembled is also unclear, though we speculate that RNA helicases may play a prominent role given their RNP-remodeling roles in other RNA biology processes and at least one prior example of precedent (RNA scaffolds and decoys enable dynamic and condition-specific responses to a variety of cellular stimuli , with lnrecedent .A unique feature of mRNAs is their potential to scaffold nascent protein interactions by virtue of the fact they also serve as protein synthesis templates. To date, characterized cytoplasmic mRNA scaffolds are driven by the presence of unique 3′UTR sequences generated by alternative cleavage and polyadenylation or paralogous gene transcription. Distinct mRNA 3′UTR sequences, with binding sites for nascent protein interaction partners can then help define a nascent protein’s interaction fate and function. How widespread this mode of facilitating protein-protein interaction is remains unknown, though it offers several potential advantages. For instance, protein interaction partners and their respective template mRNAs do not have to be as carefully orchestrated for simultaneously translation and co-localization to avoid competing off-target interactions. A nascent protein’s interaction fate could also be pre-determined by loading of its mRNA’s 3′UTR, possibly in the nucleus, with suitable nascent protein interaction partners. Additionally, this approach may facilitate generation of several distinct nascent protein subcomplexes, as occurs with BIRC3 . A caveaIdentifying new RNA scaffolds and decoys remains a significant challenge. Nonetheless, new advances in sequencing, bioinformatics and protein-RNA interaction methods promise progress toward this goal. Given the intrinsic advantages of RNA as a scaffolding molecule, and that many RNA scaffolds incorporate other functions (e.g., catalysis, modifying protein activities, guide and template functions; NF and JRB conceived and wrote the review and generated the figures and table. Both authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


By deep sequencing, we show that the two repeats give rise to an 8-nt 5′ handle and a 22-nt 3′ handle, respectively, i.e., the conserved elements of a canonical CRISPR RNA, indicating they both retained critical nucleotides for Cas6 processing during divergent degeneration. We also uncovered a minimal CreT toxin that sequesters the rare transfer RNA for isoleucine, tRNAIleCAU, with a six-codon open reading frame containing two consecutive AUA codons. To fully relieve its toxicity, both tRNAIleCAU overexpression and supply of extra agmatine (modifies the wobble base of tRNAIleCAU to decipher AUA codons) are required. By replacing AUA to AGA/AGG codons, we reprogrammed this toxin to sequester rare arginine tRNAs. These data provide essential information on CreTA origin and for future CreTA prediction, and enrich the knowledge of tRNA-sequestering small RNAs that are employed by CRISPR-Cas to get addictive to the host.Aside from providing adaptive immunity, type I CRISPR-Cas was recently unearthed to employ a noncanonical RNA guide (CreA) to transcriptionally repress an RNA toxin (CreT). Here, we report that, for most archaeal and bacterial CreTA modules, the  Adaptive immunity in prokaryotes is mediated by CRISPR-Cas systems that defend archaea and bacteria against recurrent invasions of foreign genetic elements, such as viruses and plasmids . CRISPR A complex of Cas1, Cas2 and, in some cases, also Cas4 mediates the acquisition of new spacers from an invading DNA, a process that is known as CRISPR adaptation ,6. TransTo attack a target DNA, the CRISPR effector first recognizes a conserved protospacer adjacent motif (PAM), which is critical for self versus non-self discrimination ,13. HoweHaloarcula hispanica CreTA, which consists of two RNA components, CreT and CreA. CreT is a small toxic RNA that carries a four-codon open reading frame (ORF) with two consecutive minor arginine codons (AGA) and arrests cellular growth by sequestering the cognate, rare transfer RNA (tRNAArgUCU). H. hispanica CreA is a variant of crRNA that lacks the canonical 3′ handle  and directs Cascade to suppress toxin expression based on its partial complementarity to the promoter of the toxin gene. These insights into the modulation of H. hispanica CreTA by CRISPR-Cas are critical for our understanding of the multifunctionality of CRISPR-Cas and its evolutionary and functional entanglement with toxin-antitoxin (TA) modules. However, because both the toxin and the antitoxin components are small RNAs, CreTA modules are extremely diversified and poorly conserved in sequence, which largely impedes the systematic bioinformatic analysis of their distribution and, particularly, the homology-based prediction of their toxin genes.Our recent study unearthed a diverse set of CRISPR-regulated toxin-antitoxin (CreTA) RNA pairs, which safeguard Cascade complexes, the multi-subunit effectors of type I CRISPR-Cas, by making them addictive to the host cell . In thatHalobacterium hubeiense strains, and uncover that H. hubeiense CreA is structurally more closely similar to a canonical crRNA than the H. hispanica CreA and carries two flanking repeat-derived handles. However, the flanking repeats of creA are highly divergent and degenerated in sequence, which hindered their interpretation as a minimal CRISPR array. Interestingly, we found that two highly divergent, degenerated repeats seem to be a common feature for most archaeal and bacterial creA genes, suggesting their origin and degeneration from a minimal CRISPR structure. By dissecting the elements and mechanism of H. hubeiense CreT, we also uncover a group of minimal RNA toxins that specifically sequester the rare isoleucine tRNA. By comparative analysis of H. hubeiense and H. hispanica CreT toxins, we characterize their convergent strategy to sequester a specific rare tRNA and their dependence on efficient translation signals. These data provide essential information on the origin of CreTA and for future CreTA prediction, and offers more insights into the cryptic small RNAs that associate and co-evolve with CRISPR-Cas.Here, we experimentally characterize another CreTA module that frequently associates with the type I-B CRISPR-Cas in H. hispanica strains  and uracil was added to a concentration of 50 mg/l. The strains carrying the pWL502 derivatives were grown in the modified AS-168 medium without yeast extract. Agmatine was added to a final concentration of 570 mg/l, when specified.ain DF60 ) used inain DF60  were culEscherichia coli JM109 was cultivated at 37°C in Luria-Bertani medium and used as host strain for plasmid engineering. When needed, ampicillin was supplemented to a final concentration of 100 mg/l.H. hubeiense CreTA locus . The yeast extract-subtracted AS-168 was used as the selective medium. The log-transformed data of transformation efficiency (CFU/μg) were used to calculate the average and the standard deviation, and also to perform the two-tailed Student's t test.The primers that were used in this study are listed in H. hispanica cells encoding (cas6+) or lacking (cas6-) Cas6 were transformed with pTA-tRNA. Colonies were randomly selected, separately inoculated into 10 ml of yeast extract-subtracted AS-168 medium (containing 570 mg/l agmatine), and then cultured for 4 days. After sub-inoculation and another 2-day culturing, the exponential-phase cells were collected by centrifugation and the total RNA was extracted using the TRIzol reagent  according to the standard guidelines. RNA concentration was determined using a Nanodrop 1000 spectrophotometer . A total of 50 μg of RNA was successively treated with RNA pyrophosphohydrolase and T4 polynucleotide kinase [both purchased from New England Biolabs ] according to the manufacturer's protocols. The pretreated RNA was purified using the phenol:chloroform method, followed by precipitation with the same volume of isopropanol and 0.1 volume of 3 M sodium acetate. The RNA quality was analyzed using Nanodrop 2000  prior to constructing the RNA-Seq library. Small RNA libraries were constructed with RNA molecules ranging from 30 to 300 nt, following the guideline of the NEXTFLEX Small RNA-Seq Kit , and then subjected to 150-bp paired-end sequencing on an Illumina HiSeq X Ten. The raw reads were trimmed to remove adapters and low-quality reads. A custom Perl script was finally used to map the resulting reads to the creTA sequence . RNA samples, the Century-Plus RNA ladder , and the biotin-labeled single-stranded DNA (serving as a custom size marker) were separated on an 8% polyacrylamide gel (7.6 M urea). Electrophoresis was performed in 1× TBE buffer at 200 V for approximately 1 h. The lane of the commercial RNA ladder was excised, stained by ethidium bromide, and then imaged. The remnant RNA samples and custom markers were transferred onto Biodyne B nylon membrane  by electroblotting and then UV-crosslinked to the membrane. The membrane was hybridized with a biotin-labeled probe for approximately 12 h, and the signals were visualized using the Chemiluminescent Nucleic Acid Detection Module Kit  according to the manufacturer's protocol.creTA. For each H. hispanica transformant (with the gfp-carrying plasmids or the empty vector), three individual colonies were randomly selected and separately inoculated with 10 ml of yeast extract-subtracted AS-168 medium. After cultivation to the late exponential phase, the cell culture was sampled and fluorescence was measured. Fluorescence intensity and OD600 were simultaneously determined using a microplate reader , and their ratio was used for plotting.The gene of a soluble-modified red-shifted GFP protein  was usedH. hispanica were downloaded from NCBI  and the usage frequency of Ile codons was calculated. RNAfold webserver  sequence, of which the first 1–5 and 7–11 nucleotides base pair to a DNA sequence upstream of creT, and notably, the target sequence (protospacer) is flanked by 5′-TTC-3′, which is the PAM motif of type I-B CRISPR-Cas  and ends with a thymine-rich transcription terminator (H. hubeiense ΨS appears to be sandwiched by two CRISPR repeat-like sequences (30 nt each), denoted as ΨR1 and ΨR2, respectively  transformed H. hispanica cells with a markedly reduced efficiency (∼104-fold) compared to the empty vector , and transformed H. hispanicacas6+ and cas6- strains (note that the native creTA was pre-deleted from both strains) , but transformed cas6- cells with a markedly reduced efficiency (∼104-fold). By contrast, pT-Hhub that carries only the creT gene showed very low efficiency (∼10 CFU/μg) in transforming both cas6+ and cas6- cells  in cells lacking any or all of the cascade genes, but not in those lacking cas1, cas2, cas3 or cas4  Figure . We founs Figure . It was  or cas4 . We concH. hubeiense creTA in the H. hispanica cas6+ or cas6- cells using small RNA sequencing (sRNA-seq) . Furthermore, the RNA samples were pretreated by polynucleotide kinase and 5′ pyrophosphohydrolase to activate the 5′ terminus of mature CreA (hydroxylated) and that of nascent creTA transcripts (triphosphorylated), respectively . Sequencing of the cas6- RNA samples revealed the strong transcription start site (TSS) of the creTA operon , abundant transcripts extended and gradually decreased along the creTA operon, with a prominent transcription termination site (TTS) appearing downstream of creA . Although a constitutive promoter (PphaR)  Figure . For thin Figure , upstrean Figure    was emplt Figure . TherefoH. hubeiense creA gene has a second CRISPR repeat-like sequence (ΨR2) that was significantly different from the first one (ΨR1) but also processed by the Cas6 nuclease, we wondered whether more creTA modules actually also carry two highly divergent flanking ‘repeats’, instead of one. We reanalyzed the sequence of another four haloarchaeal creTA modules predicted in our previous study .Our previous study also predicted several bacterial CreTA modules . We furtch other . TherefoH. hubeiense creTA, the abundant creT transcripts observed in the cas6- samples dramatically decreased (by >30-fold) in the cas6+ samples , generating pT-gfp and pTA-gfp, and monitored their fluorescence in H. hispanica cells (creA) produced fluorescence of equivalent intensity in cas6+ and cas6- cells. Such intensive fluorescence was also observed for pTA-gfp (carrying creA) in cas6- cells, but in cas6+ cells (where CreA was matured by Cas6), fluorescence decreased by ∼26-fold. We then introduced pT-gfp and pTA-gfp separately into cells lacking cas1, cas2, cas3, cas4 or other cascade genes, and monitored their fluorescence. The fluorescence from pT-gfp was higher than from pTA-gfp in cells lacking cas1, cas2, cas3 or cas4, and by contrast, equivalent fluorescence was produced from the two plasmids in cells lacking one or all of the cascade genes  in both cas6- and cas6+cells. By contrast, the complementarily mutated creA restored high transformation efficiency (∼105 CFU/μg). Therefore, the regulatory role of CreA depends on its limited but critical ‘seed complementarity’ to PcreTA. Interestingly, H. hispanica CreTA (H. hubeiense CreTA evolved exactly the same seed complementarity (at nucleotides 1–5 and 7–11) to achieve CreA-guided transcriptional regulation of creT.This regulation is in accord with the observation that the first 1–5 and 7–11 nucleotides (seed) of CreA ΨS base pair to the identified Pt Figure . We const Figure . When tht Figure ) was mutd Figure . We therd Figure  form theca CreTA  and H. hH. hubeiense CreT caused toxicity in H. hispanica cells. Our previous study on H. hispanica CreT demonstrated that this RNA toxin acts by sequestering tRNAArgUCU that deciphers the rare AGA codons  that have the potential to fold into a stable stem-loop structure    , which can lead to the sequestration of its cognate tRNA.Then, we asked how A codons . A strone Figure . We trun CFU/μg) . Similar CFU/μg) . When th CFU/μg) . Both th CFU/μg) . By anal) Figure . Assumin) Figure , suggest) Figure . TherefoIleCAU, and this process strictly relies on the modification of the first (wobble) position of the anticodon CAU (IleCAU is modified to 2-agmatinylcytidine (agm2C)  under the control of a strong promoter, thus generating the plasmid pTA-tRNA  than pTA-Hhub. However, compared to the empty vector (pWL502), there was still a ∼10-fold reduction in transformation efficiency, and notably, its transformants formed much smaller colonies  Figure , which a) Figure . We modiA Figure . When trs Figure . Thus, os Figure . These dH. hispanica and H. hubeiense CreT toxins share little sequence similarity, they both arrest cellular growth by sequestering rare tRNA species that decode minor codons. H. hispanica CreT sequesters tRNAArgUCU with two consecutive AGA codons, whereas H. hubeiense CreT sequesters tRNAIleCAU with two AUA codons. We sought to determine whether H. hubeiense CreT could be engineered to sequester the rare arginine tRNA species. We modified pTA-Hhub by replacing its two AUA codons with the rare AGA or AGG arginine codons  that is critical for its toxicity  RNA pairs, which safeguard the genetic integrity of the multi-subunit CRISPR effector (mainly type I) and hence protect the adaptive immunity . This prH. hispanica type I-B CRISPR-Cas  sequence, the H. hubeiense antitoxin gene contains two ΨR sequences (ΨR1 and ΨR2) flanking the ΨS sequence. Accordingly, the mature H. hispanica CreA contains only the conserved 5′ handle, whereas the mature H. hubeiense CreA contains both the 5′ handle and the 3′ handle. By sequence similarity search, we found homologs of H. hispanica and H. hubeiense creTA in 4 and 12 haloarchaeal strains, respectively array. We also noted that, in most of the two-ΨR creA genes, the last 8 nucleotides of ΨR1 comprising the 5′ handle are more similar to the co-occurring CRISPR repeat than the respective nucleotides of ΨR2. Conversely, the first 22 nucleotides of ΨR2 comprising the 3′ handle are usually more similar to the CRISPR repeat than the corresponding portion of ΨR1  during ispanica . The divdynamics .creA genes are more popular. We suppose that the two-handle CreA RNA molecules could be more stable, because the 3′ handle, which is tightly bound by Cas6 after cleavage , suggesting distinct toxicity mechanisms.F Figure . Both miis study  showed tnse CreT . When UGcreTA modules that convergently sequester a specific rare tRNA, allowing us to infer some general and specific features of their toxins and antitoxins. The finding of the present work that creA more commonly carries two CRISPR repeat-like sequences is important for understanding the origin of CreTA from CRISPR repeats and the secondary, regulatory roles of Cas proteins. With the current, limited knowledge on the tRNA-sequestering small RNAs, it is difficult to predict the toxicity mechanism for other creTA modules, which highlights the diversity of CRISPR-regulated toxins. Structural and functional dissection of additional creTA modules can be expected to further enrich our knowledge of CRISPR biology and, particularly, the addictive properties of CRISPR-Cas mediated by associated toxin-antitoxin modules.In this and the previous studies , we charhttps://nmdc.cn/resource/genomics/project) under the accession number NMDC10017848.The RNA sequencing data were deposited to the National Microbiology Data Center (NMDC) (gkab821_Supplemental_FileClick here for additional data file.


RNA live-cell imaging systems have been used to visualize subcellular mRNA distribution in living cells. The RNA-binding protein (RBP)-based RNA imaging system exploits specific RBP and the corresponding RNA recognition sequences to indirectly label mRNAs. Co-expression of fluorescent protein-fused RBP and target mRNA conjugated with corresponding RNA recognition sequences allows for visualizing mRNAs by confocal microscopy. To minimize the background fluorescence in the cytosol, the nuclear localization sequence has been used to sequester the RBP not bound to mRNA in the nucleus. However, strong fluorescence in the nucleus may limit the visualization of nucleus-localized RNA and sometimes may interfere in detecting fluorescence signals in the cytosol, especially in cells with low signal-to-noise ratio.We eliminated the background fluorescence in the nucleus by using the split fluorescent protein-based approach. We fused two different RBPs with the N- or C-terminus of split fluorescent proteins (FPs). Co-expression of RBPs with the target mRNA conjugated with the corresponding RNA recognition sequences can bring split FPs together to reconstitute functional FPs for visualizing target mRNAs. We optimized the system with minimal background fluorescence and used the imaging system to visualize mRNAs in living plant cells.We established a background-free RNA live-cell imaging system that provides a platform to visualize subcellular mRNA distribution in living plant cells. Most cells transport mRNAs to specific subcellular compartments for localized RNA translation. Instead of transporting a quantity of proteins, the transport of mRNA provides an efficient strategy to distribute proteins to specific subcellular compartments. In eukaryotic cells, asymmetric mRNA localization is a conserved mechanism to create translation hotspots for regulating spatial gene expression in response to environmental cues . The intIn living plant cells, direct visualizing of mRNAs by microscopy is challenging. Many approaches have been developed that allow visualizing mRNAs in living cells. Direct labeling of mRNAs can be achieved by incorporating in vitro-synthesized mRNA with fluorescein-labeled nucleotides or introducing molecular beacons into cells to hybridize with the target mRNA , 7. HoweAlternatively, RNA can be indirectly visualized by an RNA aptamer-based approach. In this system, the target mRNA is genetically modified by tagging with an RNA aptamer recognized by RNA-binding proteins (RBPs). Co-expression of fluorescent proteins (FPs) conjugated with RBPs with aptamer-tagged mRNA allows for indirect labelling of mRNA in living cells. MCP, a coat protein derived from MS2 bacteriophage with RNA-binding activity, and the stem loop structures recognized by MCP have been widely used to monitor mRNA localization in yeast, fruit fly, mammals and plants –13. To rAlthough the MCP-based approach has been successfully used to visualize the intracellular distribution of mobile and non-mobile mRNA in plants , the bacIn this study, we improved the RNA live-cell imaging system by eliminating the background GFP fluorescent in the nucleus. The application of this split FPs-based system detected differential subcellular distribution between mobile and non-mobile mRNAs. In addition, the split FPs-based system greatly improved the signal-to-noise ratio in the nucleus, which confers detection of mRNAs in the nucleus or at juxtanuclear region. Thus, our system broadens the application of RNA live-cell imaging system in plants.To develop the mRNA live-cell imaging system without background GFP fluorescence in the nucleus, we exploited the bimolecular fluorescence complementation system to reduce background GFP fluorescence derived from MCP-GFP not bound to mRNA. In this system, two different RBPs were fused with the N- or C-terminus of split FPs to reduce the fluorescence produced from RBP-FPs not bound to mRNA. The target mRNA was flanked with the corresponding RNA recognition sequences of the RBPs. The interaction between RBPs and RNA recognition sequences may bring two split FPs together to reconstitute a mature FP Fig. A.Fig. 2IN, amino acids 1–173 from Venus) or C-terminus fragment  of the split FP protein [N or FPC with MCP or PCP  or RFPHSL12, respectively  of MS2 and PP7 bacteriophages, MCP and PCP, which specifically bind to an RNA hairpin structure of MBS and PBS, respectively . MCP and protein . To idenNicotiana benthamiana by agro-infiltration and examined GFP fluorescence under a confocal laser scanning microscope at 3 days after infiltration. When NLS-containing MNLS-FPN was co-expressed with P-FPC or FPC-P, weak GFP fluorescence was detected in both the cytosol and nucleus  of Arabidopsis FD, which contains the nuclear localization sequence (NLS) [N or P-FPC to generate MNLS-FPN and PNLS-FPC, respectively.The DNA fragments of MCP and PCP were PCR-amplified with the following primers: MCP-For (5’-GGATCCATGGCTTCTAACTTTACTCAGTTCG-3’), MCP-Rev (5’-CTCGAGGTAGATGCCGGAGTTTGCTGCGATT-3’), PCP-For (5’-GGATCCATGTCCAAAACCATCGTTCTTTCGG-3’), PCP-Rev (5’-CTCGAGACGGCCCAGCGGCACAAGGTTGACG-3’). The DNA fragments of MCP and PCP were confirmed by sequencing and cloned into split FP-containing binary vectors pVYNE, pVYNE(R), pSCYCE, or pSCYCE(R)  to generce (NLS) , was ins35S-FT or 35S-RFP to form the p1390-35S-FTHSL12 or p1390-35S-RFPHSL12 construct, respectively.For the 12 copies of the MBS-PBS hybrid stem loop sequence (HSL12), the Pcr4-12X-MBS-PBS  DNA was Agrobacterium tumefaciens strain AGL1 carrying individual constructs was cultured in 20 mL LB broth  containing 50 μg mL−1 kanamycin, 10 mM MES, pH 5.7 and 20 μM acetosyringone at 28 °C with 220 rpm shaking for 2 days. Bacteria were pelleted by centrifugation and resuspended in infiltration medium  to OD600 = 1.0. The bacterial solution was placed at room temperature for at least 1 h. For agro-infiltration, the bacteria solution was infiltrated into the abaxial side of the 4th or 5th leaves of 3-week-old N. benthamiana by using a needle-removed 5-mL syringe. For co-infiltration with 2 or 3 different constructs, different bacterial solution or infiltration media were mixed at a 1:1 volume ratio and infiltrated into leaves. The infiltrated plants were cultured in growth chambers for 3 days, then infiltrated leaves were excised for confocal laser scanning microscopy.GFP fluorescence was observed by confocal laser scanning microscopy  with the argon laser set to 488/500–530 for excitation/emission.


The selected variant binds to the cognate RNA with a similar overall affinity although it is less structured in the unbound form than the wild-type protein domain. The variant complex association and dissociation are both slower than for the wild-type, implying different mechanistic processes involved. The profile of the wild-type and mutant complex stabilities along with molecular dynamics simulations uncovers qualitative differences in the interaction modes. In the absence of positively charged and aromatic residues, the mutant uL11 domain uses ion bridging (K+/Mg+2) interactions between the RNA sugar-phosphate backbone and glutamic acid residues as an alternative source of stabilization. This study presents experimental support to provide a new perspective on how early protein–RNA interactions evolved, where the lack of aromatic/basic residues may have been compensated by acidic residues plus metal ions.RNA–peptide/protein interactions have been of utmost importance to life since its earliest forms, reaching even before the last universal common ancestor (LUCA). However, the ancient molecular mechanisms behind this key biological interaction remain enigmatic because extant RNA–protein interactions rely heavily on positively charged and aromatic amino acids that were absent (or heavily under-represented) in the early pre-LUCA evolutionary period. Here, an RNA-binding variant of the ribosomal uL11 C-terminal domain was selected from an approximately 10 Protein–RNA complexes are ubiquitous in modern life and are essential to many stages of the cell cycle and metabolism. The abundance of ribonucleoprotein complexes generally increases with organism complexity . HoweverSome of today’s proteins and RNAs can act as independent functional and structural entities and their interaction is often transient. On the other hand, early oligopeptides and RNA are thought to have served as mutual cofactors and scaffolds, necessitating strong interactions . StructuCurrently, there are two major hypotheses concerning the nature of early peptide-RNA binding. The first scenario holds that cationic amino acids were indispensable and their biosynthesis preceded the evolution of protein–RNA interaction . Some stTo test whether strong protein–RNA interactions are conceivable in the absence of positively charged and aromatic amino acids, we performed a high-throughput variant analysis of a highly conserved RNA-binding domain of the ribosomal protein uL11. We designed combinatorial libraries that comprised: 1) the prebiotically plausible subset of 10 and 2) 14 “mid-stage” canonical amino acids, and selected RNA-binding mutants using mRNA display. Detailed characterization of viable variants from each of the two libraries unambiguously support that strong and specific protein–RNA interaction is possible in the absence of cationic and aromatic amino acids with the aid of metal ions. This mechanism of interaction confirms the feasibility of early RNA–protein interaction and brings attention to alternative RNA-protein engineering strategies.Bacillus stearothermophilus ribosomal protein uL11 (CL11) and a 58-nt domain of the large subunit ribosomal RNA (58rRNA) fragment as a selection target  were selected for detailed characterization  online. Escherichia coli and purified to homogeneity. The protein samples were used to estimate the secondary structure content by circular dichroism (CD) (Synthetic genes of the CL11 wild-type protein and CL11-E and CL11-M variants were expressed in ism (CD) . The CL1ism (CD)  online. KD constant of the CL11-E variant is about one order of magnitude smaller than those of the CL11-M variant and the wild-type CL11 . The different kon and koff values indicate different kinetics of the RNA binding of these variants (The binding propensity of the purified proteins to 58rRNA was confirmed using the electrophoretic mobility shift assay (EMSA). The wild-type sequence (CL11), as well as the selected variants CL11-E and CL11-M, exhibited an electrophoretic shift on a native PAGE gel in the presence of fluorescent 58rRNA (f58rRNA) B. Dissocvariants .The structural properties of the unbound versus 58rRNA-bound CL11 variants were inspected using mild Lon protease and RNase A treatment C and D. +2 and K+ ions. To verify the effect of metal ion concentration on the CL11-E–RNA complex, all the variant complexes were exposed to mild EDTA treatment  ion sites were observed to bridge the protein and the RNA but only in the simulation of the CL11-E–58rRNA complex. Four K+ ions were attracted to the areas of high electrostatic potential, that is, close to carboxylates of protein Glu side chains and to the phosphates of RNA backbone, but were in constant exchange between contact pairs and bulk solvent. Four presumed K+ sites were probed by manual replacement of K+ with Mg2+. Upon starting another 2 µs MD from this model, we observed stable occupation of these sites. Mg2+ ions were coordinated either directly by the 58rRNA (U28 in Mg1 site) or the protein side chains  or indirectly via water bridges  (2+ ions mediate unique CL11-E specific interactions with 58rRNA.Using the previously published X-ray structure of the CL11–58rRNA complex (PDB code 1HC8), an initial model for CL11-E was prepared by generating the appropriate mutations in PyMol. The wild-type and the CL11-E complexes with 58rRNA were prepared for molecular dynamics (MD) simulations to compare the RNA–protein interface interactions. Throughout 2 µs MD, the behavior of the proteins was relatively stable with average root-mean-square deviations of protein backbone atoms in secondary structure elements  of 0.6 and 1.1 Å, for CL11–58rRNA and CL11-E–58rRNA complexes, respectively. There were minor rearrangements around 1.3 µs and in the first 500 ns of the CL11–58rRNA and CL11-E–58rRNA complexes, respectively  online. ctively) B. It shoOur experiments show that protein binders of RNA can be found in sequences composed of a significantly reduced set of canonical amino acid alphabet. Using mRNA display, we selected rRNA binders from variant libraries of the ribosomal uL11 C-terminal domain that was composed of: 1) the prebiotically plausible subset “E” of 10 canonical amino acids (lacking both positively charged and aromatic residues) as well as 2) a reduced evolutionary mid-stage “M” alphabet of 14 amino acids .2+ or Fe2+) has been raised , even though the different kinetics (in case of CL11-E) of the binding suggests additional folding of the proteins upon binding and/or a different mode of interaction. Additional folding is further supported by the proteolytic resistance of the protein and 58rRNA species when complexed, in contrast with their proteolytic susceptibility in the free forms in case of the -M and -E variants  were designed as follows. The E library is composed only of the “early” amino acids , whereas the M library includes “mid-stage” amino acids in addition . All the remaining residues in the CL11 sequence have been randomized for the above listed amino acids using the following logic. The substitution pools at each position were selected based on: 1) environment-specific amino acid substitution tables that have been developed on the basis of acceptability of mutations within folded proteins by comparative analysis of homologous proteins  Lib tool .2, and 175 mM KCl at pH 7.9) for 1 h at room temperature. Gels were visualized by fluorescence in Typhoon FLA 9500.Protein–RNA complexes were analyzed following Xing et al. protocol . Fluoreskon, koff, and KD values were calculated from Kinetic evaluation using OneToOne fitting model.All SPR measurements were performed on a four-channel SPR sensor platform (PLASMON IV) developed at the Institute of Photonics and Electronics (IPE) of the Academy of Sciences of the Czech Republic, Prague. Gold SPR chips were functionalized following the Neburkova et al. protocol . Afterwa2, 2 mM ATP, pH 8.35). After 1 h incubation at room temperature, Lon protease (266 µM) was added to the reaction. Each condition was carried out at 37°C for 3 h with aliquots withdrawn every 60 min. Reaction products were analyzed by 16% SDS–PAGE.Lon protease was expressed and purified according to f58rRNA (1.5 µM) was incubated with or without CL11 (10 µM), CL11-M (10 µM), and CL11-E (80 µM) in buffer R with 20 mM EDTA or Lon buffer. After 2 h incubation at room temperature, commercial RNAse A (NEB) (3.3 ng/ml) was added to the reaction. Each condition was carried out at 30 or 37°C for 1 h with aliquots withdrawn every 15 min. The reactions were stopped by adding 10 µg of Proteinase K (NEB) and incubated for 5 min at 37°C. Reaction products were analyzed on 10% Mini-PROTEAN TBE-Urea Gel (BIO-RAD). Gels were visualized by fluorescence in Typhoon FLA 9500.2, 0.05% Triton X-100, pH 3.5 or 10.5, and buffer R. In the salt stability experiment, 5 mg/ml of functionalized beads were incubated for 1 h at room temperature in: buffer R, buffer R without KCl, buffer R without MgCl2, buffer R with 40 mM CaCl2 instead of MgCl2 and buffer R without KCl and MgCl2. Elution samples were loaded on 16% SDS–PAGE gel and analyzed by Western blot using Monoclonal Anti-6X His tag antibody produced in mouse conjugated with HorseRadish Peroxidase, HRP (Sigma), detected with a Immobilon Forte Western HRP substrate (Merck) and visualized by Amersham Imager 600.Magnetic Dynabeads MyOne Streptavidin C1 (Invitrogen) and Magnetic Dynabeads M-280 Tosylactivated (Invitrogen) functionalized with Neutravidin (Invitrogen) were used to immobilize 1 μM of biotinylated 58rRNA according to the manufacturer’s recommendation. The functionalized beads were washed and then incubated for 1 h with 1 μM solution of different protein variants in Buffer R + 0.05% Triton X-100 at room temperature under gentle rotation. 20 μl (5 mg/ml) beads retaining the formed complex were washed and resuspended in buffer R + 0.05% Triton X-100 at the same initial concentration and incubated for 10 min at different ranges of temperature (20–80°C). In the pH stability experiment, the beads (5 mg/ml) were incubated for 1 h at room temperature at buffers with three different pH values: 50 mM glycine, 175 mM KCl, 20 mM MgCl2+ ions except two , 2) other two Mg2+ ions which were put in place of two Os3+ for computational simplicity, and 3) one K+ ion. Water molecules were added extending 12 Å from the solute. Mg2+ and Cl− ions were added to a final concentration of 20 mM and further K+ and Cl− ions were added to a final concentration of 175 mM. Mg2+ parameters of + versus Mg2+ ions in the sites, we manually exchanged four K+ ions in the sites with four Mg2+ and started a new unrestrained MD run. The SHAKE algorithm was used to restrain all bond vibrations and hydrogen mass repartitioning to 3 Da allowed us to apply a time step of 4 fs. All the analyses were done in the CPPTRAJ program  , the CL1 program .Molecular Biology and Evolution online.msac032_Supplementary_DataClick here for additional data file.