Distinct structural bases for sequence-specific DNA binding by mammalian BEN domain proteins.

The BEN domain is a recently recognized DNA binding module that is present in diverse metazoans and certain viruses. Several BEN domain factors are known as transcriptional repressors, but, overall, relatively little is known of how BEN factors identify their targets in humans. In particular, X-ray structures of BEN domain:DNA complexes are only known for Drosophila factors bearing a single BEN domain, which lack direct vertebrate orthologs. Here, we characterize several mammalian BEN domain (BD) factors, including from two NACC family BTB-BEN proteins and from BEND3, which has four BDs. In vitro selection data revealed sequence-specific binding activities of isolated BEN domains from all of these factors. We conducted detailed functional, genomic, and structural studies of BEND3. We show that BD4 is a major determinant for in vivo association and repression of endogenous BEND3 targets. We obtained a high-resolution structure of BEND3-BD4 bound to its preferred binding site, which reveals how BEND3 identifies cognate DNA targets and shows differences with one of its non-DNA-binding BEN domains (BD1). Finally, comparison with our previous invertebrate BEN structures, along with additional structural predictions using AlphaFold2 and RoseTTAFold, reveal distinct strategies for target DNA recognition by different types of BEN domain proteins. Together, these studies expand the DNA recognition activities of BEN factors and provide structural insights into sequence-specific DNA binding by mammalian BEN proteins.

Large Stokes shift fluorescent RNAs for dual-emission fluorescence and bioluminescence imaging in live cells.

Fluorescent RNAs, aptamers that bind and activate small fluorogenic dyes, have provided a particularly attractive approach to visualizing RNAs in live cells. However, the simultaneous imaging of multiple RNAs remains challenging due to a lack of bright and stable fluorescent RNAs with bio-orthogonality and suitable spectral properties. Here, we develop the Clivias, a series of small, monomeric and stable orange-to-red fluorescent RNAs with large Stokes shifts of up to 108 nm, enabling the simple and robust imaging of RNA with minimal perturbation of the target RNA's localization and functionality. In combination with Pepper fluorescent RNAs, the Clivias enable the single-excitation two-emission dual-color imaging of cellular RNAs and genomic loci. Clivias can also be used to detect RNA-protein interactions by bioluminescent imaging both in live cells and in vivo. We believe that these large Stokes shift fluorescent RNAs will be useful tools for the tracking and quantification of multiple RNAs in diverse biological processes.

Structural basis of a small monomeric Clivia fluorogenic RNA with a large Stokes shift.

RNA-based fluorogenic modules have revolutionized the spatiotemporal localization of RNA molecules. Recently, a fluorophore named 5-((Z)-4-((2-hydroxyethyl)(methyl)amino)benzylidene)-3-methyl-2-((E)-styryl)-3,5-dihydro-4H-imidazol-4-one (NBSI), emitting in red spectrum, and its cognate aptamer named Clivia were identified, exhibiting a large Stokes shift. To explore the underlying molecular basis of this unique RNA-fluorophore complex, we determined the tertiary structure of Clivia-NBSI. The overall structure uses a monomeric, non-G-quadruplex compact coaxial architecture, with NBSI sandwiched at the core junction. Structure-based fluorophore recognition pattern analysis, combined with fluorescence assays, enables the orthogonal use of Clivia-NBSI and other fluorogenic aptamers, paving the way for both dual-emission fluorescence and bioluminescence imaging of RNA molecules within living cells. Furthermore, on the basis of the structure-based substitution assay, we developed a multivalent Clivia fluorogenic aptamer containing multiple minimal NBSI-binding modules. This innovative design notably enhances the recognition sensitivity of fluorophores both in vitro and in vivo, shedding light on future efficient applications in various biomedical and research contexts.

Structure-based characterization and compound identification of the wild-type THF class-II riboswitch.

Riboswitches are conserved regulatory RNA elements participating in various metabolic pathways. Recently, a novel RNA motif known as the folE RNA motif was discovered upstream of folE genes. It specifically senses tetrahydrofolate (THF) and is therefore termed THF-II riboswitch. To unravel the ligand recognition mechanism of this newly discovered riboswitch and decipher the underlying principles governing its tertiary folding, we determined both the free-form and bound-form THF-II riboswitch in the wild-type sequences. Combining structural information and isothermal titration calorimetry (ITC) binding assays on structure-based mutants, we successfully elucidated the significant long-range interactions governing the function of THF-II riboswitch and identified additional compounds, including alternative natural metabolites and potential lead compounds for drug discovery, that interact with THF-II riboswitch. Our structural research on the ligand recognition mechanism of the THF-II riboswitch not only paves the way for identification of compounds targeting riboswitches, but also facilitates the exploration of THF analogs in diverse biological contexts or for therapeutic applications.

Structure-based investigations of the NAD+-II riboswitch.

Riboswitches are conserved non-coding domains in bacterial mRNA with gene regulation function that are essential for maintaining enzyme co-factor metabolism. Recently, the pnuC RNA motif was reported to selectively bind nicotinamide adenine dinucleotide (NAD+), defining a novel class of NAD+ riboswitches (NAD+-II) according to phylogenetic analysis. To reveal the three-dimensional architecture and the ligand-binding mode of this riboswitch, we solved the crystal structure of NAD+-II riboswitch in complex with NAD+. Strikingly and in contrast to class-I riboswitches that form a tight recognition pocket for the adenosine diphosphate (ADP) moiety of NAD+, the class-II riboswitches form a binding pocket for the nicotinamide mononucleotide (NMN) portion of NAD+ and display only unspecific interactions with the adenosine. We support this finding by an additional structure of the class-II RNA in complex with NMN alone. The structures define a novel RNA tertiary fold that was further confirmed by mutational analysis in combination with isothermal titration calorimetry (ITC), and 2-aminopurine-based fluorescence spectroscopic folding studies. Furthermore, we truncated the pnuC RNA motif to a short RNA helical scaffold with binding affinity comparable to the wild-type motif to allude to the potential of engineering the NAD+-II motif for biotechnological applications.

Structure-based investigation of fluorogenic Pepper aptamer.

Pepper fluorescent RNAs are a recently reported bright, stable and multicolor fluorogenic aptamer tag that enable imaging of diverse RNAs in live cells. To investigate the molecular basis of the superior properties of Pepper, we determined the structures of complexes of Pepper aptamer bound with its cognate HBC or HBC-like fluorophores at high resolution by X-ray crystallography. The Pepper aptamer folds in a monomeric non-G-quadruplex tuning-fork-like architecture composed of a helix and one protruded junction region. The near-planar fluorophore molecule intercalates in the middle of the structure and is sandwiched between one non-G-quadruplex base quadruple and one noncanonical G·U wobble helical base pair. In addition, structure-based mutational analysis is evaluated by in vitro and live-cell fluorogenic detection. Taken together, our research provides a structural basis for demystifying the fluorescence activation mechanism of Pepper aptamer and for further improvement of its future application in RNA visualization.

Insights into xanthine riboswitch structure and metal ion-mediated ligand recognition.

Riboswitches are conserved functional domains in mRNA that mostly exist in bacteria. They regulate gene expression in response to varying concentrations of metabolites or metal ions. Recently, the NMT1 RNA motif has been identified to selectively bind xanthine and uric acid, respectively, both are involved in the metabolic pathway of purine degradation. Here, we report a crystal structure of this RNA bound to xanthine. Overall, the riboswitch exhibits a rod-like, continuously stacked fold composed of three stems and two internal junctions. The binding-pocket is determined by the highly conserved junctional sequence J1 between stem P1 and P2a, and engages a long-distance Watson-Crick base pair to junction J2. Xanthine inserts between a G-U pair from the major groove side and is sandwiched between base triples. Strikingly, a Mg2+ ion is inner-sphere coordinated to O6 of xanthine and a non-bridging oxygen of a backbone phosphate. Two further hydrated Mg2+ ions participate in extensive interactions between xanthine and the pocket. Our structure model is verified by ligand binding analysis to selected riboswitch mutants using isothermal titration calorimetry, and by fluorescence spectroscopic analysis of RNA folding using 2-aminopurine-modified variants. Together, our study highlights the principles of metal ion-mediated ligand recognition by the xanthine riboswitch.

Common and distinct DNA-binding and regulatory activities of the BEN-solo transcription factor family.

Recently, the BEN (BANP, E5R, and NAC1) domain was recognized as a new class of conserved DNA-binding domain. The fly genome encodes three proteins that bear only a single BEN domain ("BEN-solo" factors); namely, Insensitive (Insv), Bsg25A (Elba1), and CG9883 (Elba2). Insv homodimers preferentially bind CCAATTGG palindromes throughout the genome to mediate transcriptional repression, whereas Bsg25A and Elba2 heterotrimerize with their obligate adaptor, Elba3 (i.e., the ELBA complex), to recognize a CCAATAAG motif in the Fab-7 insulator. While these data suggest distinct DNA-binding properties of BEN-solo proteins, we performed reporter assays that indicate that both Bsg25A and Elba2 can individually recognize Insv consensus sites efficiently. We confirmed this by solving the structure of Bsg25A complexed to the Insv site, which showed that key aspects of the BEN:DNA recognition strategy are similar between these proteins. We next show that both Insv and ELBA proteins are competent to mediate transcriptional repression via Insv consensus sequences but that the ELBA complex appears to be selective for the ELBA site. Reciprocally, genome-wide analysis reveals that Insv exhibits significant cobinding to class I insulator elements, indicating that it may also contribute to insulator function. Indeed, we observed abundant Insv binding within the Hox complexes with substantial overlaps with class I insulators, many of which bear Insv consensus sites. Moreover, Insv coimmunoprecipitates with the class I insulator factor CP190. Finally, we observed that Insv harbors exclusive activity among fly BEN-solo factors with respect to regulation of Notch-mediated cell fate choices in the peripheral nervous system. This in vivo activity is recapitulated by BEND6, a mammalian BEN-solo factor that conserves the Notch corepressor function of Insv but not its capacity to bind Insv consensus sites. Altogether, our data define an array of common and distinct biochemical and functional properties of this new family of transcription factors.

RNA-Puzzles Round IV: 3D structure predictions of four ribozymes and two aptamers.

RNA-Puzzles is a collective endeavor dedicated to the advancement and improvement of RNA 3D structure prediction. With agreement from crystallographers, the RNA structures are predicted by various groups before the publication of the crystal structures. We now report the prediction of 3D structures for six RNA sequences: four nucleolytic ribozymes and two riboswitches. Systematic protocols for comparing models and crystal structures are described and analyzed. In these six puzzles, we discuss (i) the comparison between the automated web servers and human experts; (ii) the prediction of coaxial stacking; (iii) the prediction of structural details and ligand binding; (iv) the development of novel prediction methods; and (v) the potential improvements to be made. We show that correct prediction of coaxial stacking and tertiary contacts is essential for the prediction of RNA architecture, while ligand binding modes can only be predicted with low resolution and simultaneous prediction of RNA structure with accurate ligand binding still remains out of reach. All the predicted models are available for the future development of force field parameters and the improvement of comparison and assessment tools.

Structural distinctions between NAD+ riboswitch domains 1 and 2 determine differential folding and ligand binding.

Riboswitches are important gene regulatory elements frequently encountered in bacterial mRNAs. The recently discovered nadA riboswitch contains two similar, tandemly arrayed aptamer domains, with the first domain possessing high affinity for nicotinamide adenine dinucleotide (NAD+). The second domain which comprises the ribosomal binding site in a putative regulatory helix, however, has withdrawn from detection of ligand-induced structural modulation thus far, and therefore, the identity of the cognate ligand and the regulation mechanism have remained unclear. Here, we report crystal structures of both riboswitch domains, each bound to NAD+. Furthermore, we demonstrate that ligand binding to domain 2 requires significantly higher concentrations of NAD+ (or ADP retaining analogs) compared to domain 1. Using a fluorescence spectroscopic approach, we further shed light on the structural features which are responsible for the different ligand affinities, and describe the Mg2+-dependent, distinct folding and pre-organization of their binding pockets. Finally, we speculate about possible scenarios for nadA RNA gene regulation as a putative two-concentration sensor module for a time-controlled signal that is primed and stalled by the gene regulation machinery at low ligand concentrations (domain 1), and finally triggers repression of translation as soon as high ligand concentrations are reached in the cell (domain 2).

Hatchet ribozyme structure and implications for cleavage mechanism.

Small self-cleaving ribozymes catalyze site-specific cleavage of their own phosphodiester backbone with implications for viral genome replication, pre-mRNA processing, and alternative splicing. We report on the 2.1-Å crystal structure of the hatchet ribozyme product, which adopts a compact pseudosymmetric dimeric scaffold, with each monomer stabilized by long-range interactions involving highly conserved nucleotides brought into close proximity of the scissile phosphate. Strikingly, the catalytic pocket contains a cavity capable of accommodating both the modeled scissile phosphate and its flanking 5' nucleoside. The resulting modeled precatalytic conformation incorporates a splayed-apart alignment at the scissile phosphate, thereby providing structure-based insights into the in-line cleavage mechanism. We identify a guanine lining the catalytic pocket positioned to contribute to cleavage chemistry. The functional relevance of structure-based insights into hatchet ribozyme catalysis is strongly supported by cleavage assays monitoring the impact of selected nucleobase and atom-specific mutations on ribozyme activity.

The BEN domain is a novel sequence-specific DNA-binding domain conserved in neural transcriptional repressors.

We recently reported that Drosophila Insensitive (Insv) promotes sensory organ development and has activity as a nuclear corepressor for the Notch transcription factor Suppressor of Hairless [Su(H)]. Insv lacks domains of known biochemical function but contains a single BEN domain (i.e., a "BEN-solo" protein). Our chromatin immunoprecipitation (ChIP) sequencing (ChIP-seq) analysis confirmed binding of Insensitive to Su(H) target genes in the Enhancer of split gene complex [E(spl)-C]; however, de novo motif analysis revealed a novel site strongly enriched in Insv peaks (TCYAATHRGAA). We validate binding of endogenous Insv to genomic regions bearing such sites, whose associated genes are enriched for neural functions and are functionally repressed by Insv. Unexpectedly, we found that the Insv BEN domain binds specifically to this sequence motif and that Insv directly regulates transcription via this motif. We determined the crystal structure of the BEN-DNA target complex, revealing homodimeric binding of the BEN domain and extensive nucleotide contacts via α helices and a C-terminal loop. Point mutations in key DNA-contacting residues severely impair DNA binding in vitro and capacity for transcriptional regulation in vivo. We further demonstrate DNA-binding and repression activities by the mammalian neural BEN-solo protein BEND5. Altogether, we define novel DNA-binding activity in a conserved family of transcriptional repressors, opening a molecular window on this extensive gene family.

Crucial Roles of Two Hydrated Mg2+ Ions in Reaction Catalysis of the Pistol Ribozyme.

Pistol ribozymes constitute a new class of small self-cleaving RNAs. Crystal structures have been solved, providing three-dimensional snapshots along the reaction coordinate of pistol phosphodiester cleavage, corresponding to the pre-catalytic state, a vanadate mimic of the transition state, and the product. The results led to the proposed underlying chemical mechanism. Importantly, a hydrated Mg2+ ion remains innersphere-coordinated to N7 of G33 in all three states, and is consistent with its likely role as acid in general acid base catalysis (δ and ß catalysis). Strikingly, the new structures shed light on a second hydrated Mg2+ ion that approaches the scissile phosphate from its binding site in the pre-cleavage state to reach out for water-mediated hydrogen bonding in the cyclophosphate product. The major role of the second Mg2+ ion appears to be the stabilization of product conformation. This study delivers a mechanistic understanding of ribozyme-catalyzed backbone cleavage.

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

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

Pistol ribozyme adopts a pseudoknot fold facilitating site-specific in-line cleavage.

The field of small self-cleaving nucleolytic ribozymes has been invigorated by the recent discovery of the twister, twister-sister, pistol and hatchet ribozymes. We report the crystal structure of a pistol ribozyme termed env25, which adopts a compact tertiary architecture stabilized by an embedded pseudoknot fold. The G-U cleavage site adopts a splayed-apart conformation with in-line alignment of the modeled 2'-O of G for attack on the adjacent to-be-cleaved P-O5' bond. Highly conserved residues G40 (N1 position) and A32 (N3 and 2'-OH positions) are aligned to act as a general base and a general acid, respectively, to accelerate cleavage chemistry, with their roles confirmed by cleavage assays on variants, and an increased pKa of 4.7 for A32. Our structure of the pistol ribozyme defined how the overall and local topologies dictate the in-line alignment at the G-U cleavage site, with cleavage assays on variants revealing key residues that participate in acid-base-catalyzed cleavage chemistry.

Fluoride ion encapsulation by Mg2+ ions and phosphates in a fluoride riboswitch.

Significant advances in our understanding of RNA architecture, folding and recognition have emerged from structure-function studies on riboswitches, non-coding RNAs whose sensing domains bind small ligands and whose adjacent expression platforms contain RNA elements involved in the control of gene regulation. We now report on the ligand-bound structure of the Thermotoga petrophila fluoride riboswitch, which adopts a higher-order RNA architecture stabilized by pseudoknot and long-range reversed Watson-Crick and Hoogsteen A•U pair formation. The bound fluoride ion is encapsulated within the junctional architecture, anchored in place through direct coordination to three Mg(2+) ions, which in turn are octahedrally coordinated to water molecules and five inwardly pointing backbone phosphates. Our structure of the fluoride riboswitch in the bound state shows how RNA can form a binding pocket selective for fluoride, while discriminating against larger halide ions. The T. petrophila fluoride riboswitch probably functions in gene regulation through a transcription termination mechanism.

Atom-Specific Mutagenesis Reveals Structural and Catalytic Roles for an Active-Site Adenosine and Hydrated Mg2+ in Pistol Ribozymes.

The pistol RNA motif represents a new class of self-cleaving ribozymes of yet unknown biological function. Our recent crystal structure of a pre-catalytic state of this RNA shows guanosine G40 and adenosine A32 close to the G53-U54 cleavage site. While the N1 of G40 is within 3.4 Šof the modeled G53 2'-OH group that attacks the scissile phosphate, thus suggesting a direct role in general acid-base catalysis, the function of A32 is less clear. We present evidence from atom-specific mutagenesis that neither the N1 nor N3 base positions of A32 are involved in catalysis. By contrast, the ribose 2'-OH of A32 seems crucial for the proper positioning of G40 through a H-bond network that involves G42 as a bridging unit between A32 and G40. We also found that disruption of the inner-sphere coordination of the active-site Mg2+ cation to N7 of G33 makes the ribozyme drastically slower. A mechanistic proposal is suggested, with A32 playing a structural role and hydrated Mg2+ playing a catalytic role in cleavage.

c-di-AMP binds the ydaO riboswitch in two pseudo-symmetry-related pockets.

The ydaO riboswitch, involved in sporulation, osmotic stress responses and cell wall metabolism, targets the second messenger cyclic-di-AMP with subnanomolar affinity. We have solved the structure of c-di-AMP bound to the Thermoanaerobacter tengcongensis ydaO riboswitch, thereby identifying a five-helical scaffold containing a zippered-up bubble, a pseudoknot and long-range tertiary base pairs. Highlights include the identification of two c-di-AMP binding pockets on the same face of the riboswitch, related by pseudo-two-fold symmetry, with potential for cross-talk between sites mediated by adjacently positioned base-stacking alignments connecting pockets. The adenine rings of bound c-di-AMP molecules are wedged between bases and stabilized by stacking, base-sugar and sugar-sugar intermolecular hydrogen bonding interactions. The structural studies are complemented by isothermal titration calorimetry-based binding studies of mutants mediating key tertiary intermolecular contacts. The T. tengcongensis ydaO riboswitch, like its Bacillus subtilis counterpart, most likely functions through a transcription termination mechanism, with the c-di-AMP bound state representing an 'off' switch.

A Mini-Twister Variant and Impact of Residues/Cations on the Phosphodiester Cleavage of this Ribozyme Class.

Nucleolytic ribozymes catalyze site-specific cleavage of their phosphodiester backbones. A minimal version of the twister ribozyme is reported that lacks the phylogenetically conserved stem P1 while retaining wild-type activity. Atomic mutagenesis revealed that nitrogen atoms N1 and N3 of the adenine-6 at the cleavage site are indispensable for cleavage. By NMR spectroscopy, a pKa value of 5.1 was determined for a (13) C2-labeled adenine at this position in the twister ribozyme, which is significantly shifted compared to the pKa of the same adenine in the substrate alone. This finding pinpoints at a potential role for adenine-6 in the catalytic mechanism besides the previously identified invariant guanine-48 and a Mg(2+) ion, both of which are directly coordinated to the non-bridging oxygen atoms of the scissile phosphate; for the latter, additional evidence stems from the observation that Mn(2+) or Cd(2+) accelerated cleavage of phosphorothioate substrates. The relevance of this metal ion binding site is further emphasized by a new 2.6 ŠX-ray structure of a 2'-OCH3 -U5 modified twister ribozyme.

Structure-based insights into recognition and regulation of SAM-sensing riboswitches.

Riboswitches are highly conserved RNA elements that located in the 5'-UTR of mRNAs, which undergo real-time structure conformational change to achieve the regulation of downstream gene expression by sensing their cognate ligands. S-adenosylmethionine (SAM) is a ubiquitous methyl donor for transmethylation reactions in all living organisms. SAM riboswitch is one of the most abundant riboswitches that bind to SAM with high affinity and selectivity, serving as regulatory modules in multiple metabolic pathways. To date, seven SAM-specific riboswitch classes that belong to four families, one SAM/SAH riboswitch and one SAH riboswitch have been identified. Each SAM riboswitch family has a well-organized tertiary core scaffold to support their unique ligand-specific binding pocket. In this review, we summarize the current research progress on the distribution, structure, ligand recognition and gene regulation mechanism of these SAM-related riboswitch families, and further discuss their evolutionary prospects and potential applications.