TectoRNA: modular assembly units for the construction of RNA nano-objects

Structural information on complex biological RNA molecules can be exploited to design tectoRNAs or artificial modular RNA units that can self-assemble through tertiary interactions thereby forming nanoscale RNA objects. The selective interactions of hairpin tetraloops with their receptors can be used to mediate tectoRNA assembly. Here we report on the modulation of the specificity and the strength of tectoRNA assembly (in the nanomolar to micromolar range) by variation of the length of the RNA subunits, the nature of their interacting motifs and the degree of flexibility of linker regions incorporated into the molecules. The association is also dependent on the concentration of magnesium. Monitoring of tectoRNA assembly by lead(II) cleavage protection indicates that some degree of structural flexibility is required for optimal binding. With tectoRNAs one can compare the binding affinities of different tertiary motifs and quantify the strength of individual interactions. Furthermore, in analogy to the synthons used in organic chemistry to synthesize more complex organic compounds, tectoRNAs form the basic assembly units for constructing complex RNA structures on the nanometer scale. Thus, tectoRNA provides a means for constructing molecular scaffoldings that organize functional modules in three-dimensional space for a wide range of applications.     

Promoting RNA helical stacking via A-minor junctions

RNA molecules take advantage of prevalent structural motifs to fold and assemble into well-defined 3D architectures. The A-minor junction is a class of RNA motifs that specifically controls coaxial stacking of helices in natural RNAs. A sensitive self-assembling supra-molecular system was used as an assay to compare several natural and previously unidentified A-minor junctions by native polyacrylamide gel electrophoresis and atomic force microscopy. This class of modular motifs follows a topological rule that can accommodate a variety of interchangeable A-minor interactions with distinct local structural motifs. Overall, two different types of A-minor junctions can be distinguished based on their functional self-assembling behavior: one group makes use of triloops or GNRA and GNRA-like loops assembling with helices, while the other takes advantage of more complex tertiary receptors specific for the loop to gain higher stability. This study demonstrates how different structural motifs of RNA can contribute to the formation of topologically equivalent helical stacks. It also exemplifies the need of classifying RNA motifs based on their tertiary structural features rather than secondary structural features. The A-minor junction rule can be used to facilitate tertiary structure prediction of RNAs and rational design of RNA parts for nanobiotechnology and synthetic biology.     

The UA_handle: a versatile submotif in stable RNA architectures

Stable RNAs are modular and hierarchical 3D architectures taking advantage of recurrent structural motifs to form extensive non-covalent tertiary interactions. Sequence and atomic structure analysis has revealed a novel submotif involving a minimal set of five nucleotides, termed the UA_handle motif (5′XU/AN_nX3′). It consists of a U:A Watson–Crick: Hoogsteen trans base pair stacked over a classic Watson–Crick base pair, and a bulge of one or more nucleotides that can act as a handle for making different types of long-range interactions. This motif is one of the most versatile building blocks identified in stable RNAs. It enters into the composition of numerous recurrent motifs of greater structural complexity such as the T-loop, the 11-nt receptor, the UAA/GAN and the G-ribo motifs. Several structural principles pertaining to RNA motifs are derived from our analysis. A limited set of basic submotifs can account for the formation of most structural motifs uncovered in ribosomal and stable RNAs. Structural motifs can act as structural scaffoldings and be functionally and topologically equivalent despite sequence and structural differences. The sequence network resulting from the structural relationships shared by these RNA motifs can be used as a proto-language for assisting prediction and rational design of RNA tertiary structures.     

Nano-sandwich composite by kinetic trapping assembly from protein and nucleic acid

Design and preparation of layered composite materials alternating between nucleic acids and proteins has been elusive due to limitations in occurrence and geometry of interaction sites in natural biomolecules. We report the design and kinetically controlled stepwise synthesis of a nano-sandwich composite by programmed noncovalent association of protein, DNA and RNA modules. A homo-tetramer protein core was introduced to control the self-assembly and precise positioning of two RNA–DNA hybrid nanotriangles in a co-parallel sandwich arrangement. Kinetically favored self-assembly of the circularly closed nanostructures at the protein was driven by the intrinsic fast folding ability of RNA corner modules which were added to precursor complex of DNA bound to the protein. The 3D architecture of this first synthetic protein–RNA–DNA complex was confirmed by fluorescence labeling and cryo-electron microscopy studies. The synthesis strategy for the nano-sandwich composite provides a general blueprint for controlled noncovalent assembly of complex supramolecular architectures from protein, DNA and RNA components, which expand the design repertoire for bottom-up preparation of layered biomaterials.     

An Integrative Approach to the Study of Filamentous Oligomeric Assemblies,with Application to RecA

Oligomeric macromolecules in the cell self-organize into a wide variety of geometrical motifs such as helices, rings or linear filaments. The recombinase proteins involved in homologous recombination present many such assembly motifs. Here, we examine in particular the polymorphic characteristics of RecA, the most studied member of the recombinase family, using an integrative approach that relates local modes of monomer/monomer association to the global architecture of their screw-type organization. In our approach, local modes of association are sampled via docking or Monte Carlo simulations. This enables shedding new light on fiber morphologies that may be adopted by the RecA protein. Two distinct RecA helical morphologies, the so-called “extended” and “compressed” forms, are known to play a role in homologous recombination. We investigate the variability within each form in terms of helical parameters and steric accessibility. We also address possible helical discontinuities in RecA filaments due to multiple monomer-monomer association modes. By relating local interface organization to global filament morphology, the strategies developed here to study RecA self-assembly are particularly well suited to other DNA-binding proteins and to filamentous protein assemblies in general.     

Hydration of protein–RNA recognition sites

We investigate the role of water molecules in 89 protein–RNA complexes taken from the Protein Data Bank. Those with tRNA and single-stranded RNA are less hydrated than with duplex or ribosomal proteins. Protein–RNA interfaces are hydrated less than protein–DNA interfaces, but more than protein–protein interfaces. Majority of the waters at protein–RNA interfaces makes multiple H-bonds; however, a fraction do not make any. Those making H-bonds have preferences for the polar groups of RNA than its partner protein. The spatial distribution of waters makes interfaces with ribosomal proteins and single-stranded RNA relatively ‘dry’ than interfaces with tRNA and duplex RNA. In contrast to protein–DNA interfaces, mainly due to the presence of the 2′OH, the ribose in protein–RNA interfaces is hydrated more than the phosphate or the bases. The minor groove in protein–RNA interfaces is hydrated more than the major groove, while in protein–DNA interfaces it is reverse. The strands make the highest number of water-mediated H-bonds per unit interface area followed by the helices and the non-regular structures. The preserved waters at protein–RNA interfaces make higher number of H-bonds than the other waters. Preserved waters contribute toward the affinity in protein–RNA recognition and should be carefully treated while engineering protein–RNA interfaces.     

Molecular dynamics simulations suggest that RNA three-way junctions can act as flexible RNA structural elements in the ribosome

We present extensive explicit solvent molecular dynamics analysis of three RNA three-way junctions (3WJs) from the large ribosomal subunit: the 3WJ formed by Helices 90–92 (H90–H92) of 23S rRNA; the 3WJ formed by H42–H44 organizing the GTPase associated center (GAC) of 23S rRNA; and the 3WJ of 5S rRNA. H92 near the peptidyl transferase center binds the 3′-CCA end of amino-acylated tRNA. The GAC binds protein factors and stimulates GTP hydrolysis driving protein synthesis. The 5S rRNA binds the central protuberance and A-site finger (ASF) involved in bridges with the 30S subunit. The simulations reveal that all three 3WJs possess significant anisotropic hinge-like flexibility between their stacked stems and dynamics within the compact regions of their adjacent stems. The A-site 3WJ dynamics may facilitate accommodation of tRNA, while the 5S 3WJ flexibility appears to be essential for coordinated movements of ASF and 5S rRNA. The GAC 3WJ may support large-scale dynamics of the L7/L12-stalk region. The simulations reveal that H42–H44 rRNA segments are not fully relaxed and in the X-ray structures they are bent towards the large subunit. The bending may be related to L10 binding and is distributed between the 3WJ and the H42–H97 contact.     

Computational generation and screening of RNA motifs in large nucleotide sequence pools

Although identification of active motifs in large random sequence pools is central to RNA in vitro selection, no systematic computational equivalent of this process has yet been developed. We develop a computational approach that combines target pool generation, motif scanning and motif screening using secondary structure analysis for applications to 10¹²–10¹⁴-sequence pools; large pool sizes are made possible using program redesign and supercomputing resources. We use the new protocol to search for aptamer and ribozyme motifs in pools up to experimental pool size (10¹⁴ sequences). We show that motif scanning, structure matching and flanking sequence analysis, respectively, reduce the initial sequence pool by 6–8, 1–2 and 1 orders of magnitude, consistent with the rare occurrence of active motifs in random pools. The final yields match the theoretical yields from probability theory for simple motifs and overestimate experimental yields, which constitute lower bounds, for aptamers because screening analyses beyond secondary structure information are not considered systematically. We also show that designed pools using our nucleotide transition probability matrices can produce higher yields for RNA ligase motifs than random pools. Our methods for generating, analyzing and designing large pools can help improve RNA design via simulation of aspects of in vitro selection.     

Organization of developmental enhancers in the Drosophila embryo

Most cell-specific enhancers are thought to lack an inherent organization, with critical binding sites distributed in a more or less random fashion. However, there are examples of fixed arrangements of binding sites, such as helical phasing, that promote the formation of higher-order protein complexes on the enhancer DNA template. Here, we investigate the regulatory ‘grammar’ of nearly 100 characterized enhancers for developmental control genes active in the early Drosophila embryo. The conservation of grammar is examined in seven divergent Drosophila genomes. Linked binding sites are observed for particular combinations of binding motifs, including Bicoid–Bicoid, Hunchback–Hunchback, Bicoid–Dorsal, Bicoid–Caudal and Dorsal–Twist. Direct evidence is presented for the importance of Bicoid–Dorsal linkage in the integration of the anterior–posterior and dorsal–ventral patterning systems. Hunchback–Hunchback interactions help explain unresolved aspects of segmentation, including the differential regulation of the eve stripe 3 + 7 and stripe 4 + 6 enhancers. We also present evidence that there is an under-representation of nucleosome positioning sequences in many enhancers, raising the possibility for a subtle higher-order structure extending across certain enhancers. We conclude that grammar of gene control regions is pervasively used in the patterning of the Drosophila embryo.     

A specific role for the C-terminal region of the Poly(A)-binding protein in mRNA decay

mRNA poly(A) tails affect translation, mRNA export and mRNA stability, with translation initiation involving a direct interaction between eIF4G and the poly(A)-binding protein Pab1. The latter factor contains four RNA recognition motifs followed by a C-terminal region composed of a linker and a PABC domain. We show here that yeast mutants lacking the C-terminal domains of Pab1 display specific synthetic interactions with mutants in the 5′-3′ mRNA decay pathway. Moreover, these mutations impair mRNA decay in vivo without significantly affecting mRNA export or translation. Inhibition of mRNA decay occurs through slowed deadenylation. In vitro analyses demonstrate that removal of the Pab1 linker domain directly interferes with the ability of the Pop2–Ccr4 complex to deadenylate the Pab1-bound poly(A). Binding assays demonstrate that this results from a modulation of poly(A) packaging by the Pab1 linker region. Overall, our results demonstrate a direct involvement of Pab1 in mRNA decay and reveal the modular nature of this factor, with different domains affecting various cellular processes. These data suggest new models involving the modulation of poly(A) packaging by Pab1 to control mRNA decay.     

An intermolecular RNA triplex provides insight into structural determinants for the pseudoknot stimulator of ?1 ribosomal frameshifting

An efficient −1 programmed ribosomal frameshifting (PRF) signal requires an RNA slippery sequence and a downstream RNA stimulator, and the hairpin-type pseudoknot is the most common stimulator. However, a pseudoknot is not sufficient to promote −1 PRF. hTPK-DU177, a pseudoknot derived from human telomerase RNA, shares structural similarities with several −1 PRF pseudoknots and is used to dissect the roles of distinct structural features in the stimulator of −1 PRF. Structure-based mutagenesis on hTPK-DU177 reveals that the −1 PRF efficiency of this stimulator can be modulated by sequential removal of base–triple interactions surrounding the helical junction. Further analysis of the junction-flanking base triples indicates that specific stem–loop interactions and their relative positions to the helical junction play crucial roles for the −1 PRF activity of this pseudoknot. Intriguingly, a bimolecular pseudoknot approach based on hTPK-DU177 reveals that continuing triplex structure spanning the helical junction, lacking one of the loop-closure features embedded in pseudoknot topology, can stimulate −1 PRF. Therefore, the triplex structure is an essential determinant for the DU177 pseudoknot to stimulate −1 PRF. Furthermore, it suggests that −1 PRF, induced by an in-trans RNA via specific base–triple interactions with messenger RNAs, can be a plausible regulatory function for non-coding RNAs.     

利用激光AFM和TEM探索肌动蛋白体外自装配聚合结构

细胞内肌动蛋白(actin)通过与actin结合蛋白(actin binding proteins,ABPs)相互作用,形成以F-actin为基础多种ABPs参与装配的高度有序的超分子聚合结构,行使各种重要生理功能。在体外聚合条件下,不存在F-actin稳定剂时纯化的actin主要通过自装配形成大尺度的聚集堆积结构;这种表观无序的结构体系由于被认为不具备细胞功能活性而受到忽视。利用激光原子力显微镜(atomic force microscope,AFM)和透射电子显微镜(transmission electron microscope,TEM)技术,对actin体外通过自装配过程形成的大尺度聚集结构进行了细致的观察和分析。研究发现,actin在体外通过自装配过程除了形成无序的蛋白堆积物之外,还能够聚合形成复杂的离散结构,包括树状分支的纤维丛、无规卷曲的纤维簇以及具有不同直径的长纤维等;这些大尺度纤维复合物明显不同于在ABPs或过量F-actin稳定剂参与下形成的由单根微丝和微丝束构成的聚合结构。表明无ABPs或F-actin稳定剂存在的情况下,体外聚合的F-actin在一定条件下可进一步聚集缠绕形成复杂的纤维结构或无序的蛋白堆积物。事实上,actin自装配过程反映了其固有的聚合热力学特性,深入探索将有助于理解ABPs在体内actin超分子聚合结构体系装配中的调控作用及其分子机制。     

The interaction networks of structured RNAs

All pairwise interactions occurring between bases which could be detected in three-dimensional structures of crystallized RNA molecules are annotated on new planar diagrams. The diagrams attempt to map the underlying complex networks of base–base interactions and, especially, they aim at conveying key relationships between helical domains: co-axial stacking, bending and all Watson–Crick as well as non-Watson–Crick base pairs. Although such wiring diagrams cannot replace full stereographic images for correct spatial understanding and representation, they reveal structural similarities as well as the conserved patterns and distances between motifs which are present within the interaction networks of folded RNAs of similar or unrelated functions. Finally, the diagrams could help devising methods for meaningfully transforming RNA structures into graphs amenable to network analysis.     

Conformation and protein interactions of intramolecular DNA and phosphorothioate four-way junctions

The objectives of this study are to evaluate the structure and protein recognition features of branched DNA four-way junctions in an effort to explore the therapeutic potential of these molecules. The classic immobile DNA 4WJ, J1, is used as a matrix to design novel intramolecular junctions including natural and phosphorothioate bonds. Here we have inserted H2-type mini-hairpins into the helical termini of the arms of J1 to generate four novel intramolecular four-way junctions. Hairpins are inserted to reduce end fraying and effectively eliminate potential nuclease binding sites. We compare the structure and protein recognition features of J1 with four intramolecular four-way junctions: i-J1, i-J1(PS1), i-J1(PS2) and i-J1(PS3). Circular dichroism studies suggest that the secondary structure of each intramolecular 4WJ is composed predominantly of B-form helices. Thermal unfolding studies indicate that intramolecular four-way junctions are significantly more stable than J1. The T_m values of the hairpin four-way junctions are 25.2° to 32.2°C higher than the control, J1. With respect to protein recognition, gel shift assays reveal that the DNA-binding proteins HMGBb1 and HMGB1 bind the hairpin four-way junctions with affinity levels similar to control, J1. To evaluate nuclease resistance, four-way junctions are incubated with DNase I, exonuclease III (Exo III) and T5 exonuclease (T5 Exo). The enzymes probe nucleic acid cleavage that occurs non-specifically (DNase I) and in a 5ʹ→3ʹ (T5 Exo) and 3ʹ→5ʹ direction (Exo III). The nuclease digestion assays clearly show that the intramolecular four-way junctions possess significantly higher nuclease resistance than the control, J1.     

Measurement of the Change in Twist at a Helical Junction in RNA Using the?Orientation Dependence of FRET

Indocarbocyanine fluorophores attached via the 5′ terminus of double-stranded nucleic acids have a strong propensity to stack onto the terminal basepair. We previously demonstrated that the efficiency of fluorescence resonance energy transfer between cyanine 3 and 5 terminally attached to duplex species exhibits a pronounced modulation with helix length. This results from a systematic variation in the orientation parameter κ² as the relative rotation of the fluorophore transition moments changes due to the helical geometry. Analysis of such profiles provides a rich source of orientational information. In this work, we applied this methodology to the structure of a three-way helical junction that plays an important role in the hepatitis C virus internal ribosome entry site. By comparing matched pairs of duplex and junction species, we were able to measure the change in rotation at the junction. The data reveal a 29.5° overwinding and a small axial extension. This shows the power of this approach for measuring orientational information in biologically important RNA junctions.