RNA Hairpin Research Articles

Simultaneous NMR characterisation of multiple minima in the free energy landscape of an RNA UUCG tetraloop.

RNA molecules in solution tend to undergo structural fluctuations of relatively large amplitude and to populate a range of different conformations some of which with low populations. It is still very challenging, however, to characterise the structures of these low populated states and to understand their functional roles. In the present study, we address this problem by using NMR residual dipolar couplings (RDCs) as structural restraints in replica-averaged metadynamics (RAM) simulations. By applying this approach to a 14-mer RNA hairpin containing the prototypical UUCG tetraloop motif, we show that it is possible to construct the free energy landscape of this RNA molecule. This free energy landscapes reveals the surprisingly rich dynamics of the UUCG tetraloop and identifies the multiple substates that exist in equilibrium owing to thermal fluctuations. The approach that we present is general and can be applied to the study of the free energy landscapes of other RNA or RNA-protein systems.

Revisiting GNRA and UNCG folds: U-turns versus Z-turns in RNA hairpin loops.

When thinking about RNA three-dimensional structures, coming across GNRA and UNCG tetraloops is perceived as a boon since their folds have been extensively described. Nevertheless, analyzing loop conformations within RNA and RNP structures led us to uncover several instances of GNRA and UNCG loops that do not fold as expected. We noticed that when a GNRA does not assume its “natural” fold, it adopts the one we typically associate with a UNCG sequence. The same folding interconversion may occur for loops with UNCG sequences, for instance within tRNA anticodon loops. Hence, we show that some structured tetranucleotide sequences starting with G or U can adopt either of these folds. The underlying structural basis that defines these two fold types is the mutually exclusive stacking of a backbone oxygen on either the first (in GNRA) or the last nucleobase (in UNCG), generating an oxygen–π contact. We thereby propose to refrain from using sequences to distinguish between loop conformations. Instead, we suggest using descriptors such as U-turn (for “GNRA-type” folds) and a newly described Z-turn (for “UNCG-type” folds). Because tetraloops adopt for the largest part only two (inter)convertible turns, we are better able to interpret from a structural perspective loop interchangeability occurring in ribosomes and viral RNA. In this respect, we propose a general view on the inclination for a given sequence to adopt (or not) a specific fold. We also suggest how long-noncoding RNAs may adopt discrete but transient structures, which are therefore hard to predict.

Down-regulation of protein kinase, DNA-activated, catalytic polypeptide attenuates tumor progression and is an independent prognostic predictor of survival in prostate cancer

ObjectiveProtein kinase, DNA-activated, catalytic polypeptide (PRKDC) is a critical component of DNA repair machinery and its dysregulated expression has been observed in various cancer types or premalignant cells. However, its role in prostate cancer (PCa) development and its prognostic significance in PCa is unknown. MethodsThe mRNA and protein levels of PRKDC were analyzed in 15 pairs of PCa and benign prostatic hyperplasia tissues as well as PCa cell lines by quantitative real-time polymerase chain reaction and Western blot, respectively. Small interfering RNA and short hairpin RNA-mediated knockdown of PRKDC, followed by cell proliferation, colony formation, and soft agar assays were performed. Xenograft mouse model was used to evaluate in vivo effects of PRKDC knockdown. The association between PRKDC expression and clinicopathologic features was assessed by χ2 tests. Kaplan-Meier analysis was performed to investigate the association between PRKDC expression and overall survival. Cox proportional hazards regression models were used to examine the prognostic significance of PRKDC. ResultsExpression of PRKDC mRNA and protein was notably higher in PCa tissues and PCa cell lines. Knockdown of PRKDC markedly reduced cell proliferation, colony formation efficiency, and soft agar growth in DU145 cells. Down-regulation of PRKDC inhibited tumor growth of DU145 xenografts and enhance mice survival. In addition, PRKDC expression in PCa was significantly associated with Gleason score (P = 0.01), tumor stage (P = 0.028), and distant metastasis (P = 0.025). Patients with PCa having higher PRKDC expression had substantially shorter survival than patients with lower PRKDC expression. ConclusionDown-regulation of PRKDC attenuates tumor progression in PCa. PRKDC may potentially be a prognostic biomarker in PCa.

Exploring the effects of temperature and pressure on the structure and stability of a small RNA hairpin

RNAs perform multiple vital roles within cells, including catalyzing biological reactions and expression of proteins. Small RNA hairpins (sRNAh) are the smallest functional entities of nucleic acids and are involved in various important biological functions such as ligand binding and tertiary folding initiation of proteins. We investigated the conformational and free energy landscape of the sRNAh gcUUCGgc over a wide range of temperatures and pressures using fluorescence resonance energy transfer, Fourier-transform infrared and UV/Vis spectroscopy as well as small-angle X-ray scattering on the unlabeled and/or fluorescently labeled sRNAh. The sRNAh shows a broad melting profile with continuous increase of unpaired conformations up to about 60°C. However, the sRNAh structure might not be fully unfolded at temperatures as high as 90°C and still comprise various partially unfolded compact conformations. Pressure up to 400MPa has a small effect on the base pairing and base stacking interactions of the sRNAh, indicating small conformational perturbations, only, which might originate from minor changes in packing and hydration of the RNA molecule upon compression. Pressurization at 70°C, i.e. at a temperature above the melting transition, has no significant effect on the conformational ensemble of the sRNAh, i.e., it does not promote formation of new native stem connections after thermal denaturation. Finally, we noticed that Cy3/Cy5 labeling of the sRNAh changes, probably via stacking interactions between the fluorescent dyes and the nucleotide rings, the stability of the sRNAh, thereby rendering FRET analysis of the conformational dynamics of such small RNA structure inappropriate.

Solution structure of the 5'-terminal hairpin of the 7SK small nuclear RNA.

The small nuclear 7SK RNA regulates RNA polymerase II (RNA Pol II) transcription, by sequestering and inhibiting the positive transcription elongation factor b (P-TEFb). P-TEFb is stored in the 7SK ribonucleoprotein (RNP) that contains the three nuclear proteins Hexim1, LaRP7, and MePCE. P-TEFb interacts with the protein Hexim1 and the 7SK RNA. Once P-TEFb is released from the 7SK RNP, it activates transcription by phosphorylating the C-terminal domain of RNA Pol II. P-TEFb also plays a crucial role in the replication of the human immunodeficiency virus HIV-1, through its recruitment by the viral transactivator Tat. Previous work demonstrated that the protein Tat promotes the release of P-TEFb from the 7SK RNP through direct binding to the 7SK RNA. Hexim1 and Tat proteins both comprise conserved and similar arginine-rich motifs that were identified to bind the 7SK RNA at a repeated GAUC site located at the top of the 5′-terminal hairpin (HPI). Here, we report the solution structure of this region as determined by nuclear magnetic resonance, to identify HPI structural features recognized by Hexim1 and Tat. The HPI solution structure displays an elongated shape featuring four helical segments interrupted by one internal loop and three bulges with distinct folds. In particular, the repeated GAUC motif adopts a pre-organized geometry. Our results suggest that the binding of Hexim1 and Tat to the 7SK RNA could originate from a conformational selection of this motif, highlighting how RNA local structure could lead to an adaptive recognition of their partners.

Sequence-selective recognition of double-stranded RNA and enhanced cellular uptake of cationic nucleobase and backbone-modified peptide nucleic acids.

Sequence-selective recognition of complex RNAs in live cells could find broad applications in biology, biomedical research, and biotechnology. However, specific recognition of structured RNA is challenging, and generally applicable and effective methods are lacking. Recently, we found that peptide nucleic acids (PNAs) were unusually well-suited ligands for recognition of double-stranded RNAs. Herein, we report that 2-aminopyridine (M) modified PNAs and their conjugates with lysine and arginine tripeptides form strong (Ka = 9.4 to 17 × 107 M−1) and sequence-selective triple helices with RNA hairpins at physiological pH and salt concentration. The affinity of PNA–peptide conjugates for the matched RNA hairpins was unusually high compared to the much lower affinity for DNA hairpins of the same sequence (Ka = 0.05 to 1.1 × 107 M−1). The binding of double-stranded RNA by M-modified PNA–peptide conjugates was a relatively fast process (kon = 2.9 × 104 M−1 sec−1) compared to the notoriously slow triple helix formation by oligodeoxynucleotides (kon ∼ 103 M−1 sec−1). M-modified PNA–peptide conjugates were not cytotoxic and were efficiently delivered in the cytosol of HEK293 cells at 10 µM. Surprisingly, M-modified PNAs without peptide conjugation were also taken up by HEK293 cells, which, to the best of our knowledge, is the first example of heterocyclic base modification that enhances the cellular uptake of PNA. Our results suggest that M-modified PNA–peptide conjugates are promising probes for sequence-selective recognition of double-stranded RNA in live cells and other biological systems.

HLARP7 C-terminal domain contains an xRRM that binds the 3' hairpin of 7SK RNA.

The 7SK small nuclear ribonucleoprotein (snRNP) sequesters and inactivates the positive transcription elongation factor b (P-TEFb), an essential eukaryotic mRNA transcription factor. The human La-related protein group 7 (hLARP7) is a constitutive component of the 7SK snRNP and localizes to the 3′ terminus of the 7SK long noncoding RNA. hLARP7, and in particular its C-terminal domain (CTD), is essential for 7SK RNA stability and assembly with P-TEFb. The hLARP7 N-terminal La module binds and protects the 3′ end from degradation, but the structural and functional role of its CTD is unclear. We report the solution NMR structure of the hLARP7 CTD and show that this domain contains an xRRM, a class of atypical RRM first identified in the Tetrahymena thermophila telomerase LARP7 protein p65. The xRRM binds the 3′ end of 7SK RNA at the top of stem-loop 4 (SL4) and interacts with both unpaired and base-paired nucleotides. This study confirms that the xRRM is general to the LARP7 family of proteins and defines the binding site for hLARP7 on the 7SK RNA, providing insight into function.

Asymmetric cryo-EM structure of the canonical Allolevivirus Qβ reveals a single maturation protein and the genomic ssRNA in situ

Single-stranded (ss) RNA viruses infect all domains of life. To date, for most ssRNA virions, only the structures of the capsids and their associated protein components have been resolved to high resolution. Qβ, an ssRNA phage specific for the conjugative F-pilus, has a T = 3 icosahedral lattice of coat proteins assembled around its 4,217 nucleotides of genomic RNA (gRNA). In the mature virion, the maturation protein, A2, binds to the gRNA and is required for adsorption to the F-pilus. Here, we report the cryo-electron microscopy (cryo-EM) structures of Qβ with and without symmetry applied. The icosahedral structure, at 3.7-Å resolution, resolves loops not previously seen in the published X-ray structure, whereas the asymmetric structure, at 7-Å resolution, reveals A2 and the gRNA. A2 contains a bundle of α-helices and replaces one dimer of coat proteins at a twofold axis. The helix bundle binds gRNA, causing denser packing of RNA in its proximity, which asymmetrically expands the surrounding coat protein shell to potentially facilitate RNA release during infection. We observe a fixed pattern of gRNA organization among all viral particles, with the major and minor grooves of RNA helices clearly visible. A single layer of RNA directly contacts every copy of the coat protein, with one-third of the interactions occurring at operator-like RNA hairpins. These RNA-coat interactions stabilize the tertiary structure of gRNA within the virion, which could further provide a roadmap for capsid assembly.

Persistent RNA virus infection of lepidopteran cell lines: Interactions with the RNAi machinery

RNAi is broadly used as a technique for specific gene silencing in insects but few studies have investigated the factors that can affect its efficiency. Viral infections have the potential to interfere with RNAi through their production of viral suppressors of RNAi (VSRs) and the production of viral small RNAs that can saturate and inactivate the RNAi machinery. In this study, the impact of persistent infection of the RNA viruses Flock house virus (FHV) and Macula-like virus (MLV) on RNAi efficiency was investigated in selected lepidopteran cell lines. Lepidopteran cell lines were found to be readily infected by both viruses without any apparent pathogenic effects, with the exception of Bombyx-derived Bm5 and BmN4 cells, which could not be infected by FHV. Because Sf21 cells were free from both FHV and MLV and Hi5-SF were free from FHV and only contained low levels of MLV, they were tested to evaluate the impact of the presence of the virus. Two types of RNAi reporter assays however did not detect a significant interference with gene silencing in infected Sf21 and Hi5-SF cells when compared to virus-free cells. In Hi5 cells, the presence of FHV could be easily cleared through the expression of an RNA hairpin that targets its VSR gene, confirming that the RNAi mechanism was not inhibited. Sequencing indicated that the B2 RNAi inhibitor gene of FHV and a putative VSR gene from MLV were intact in persistently infected cell lines, indicating that protection against RNAi remains essential for virus survival. It is proposed that infection levels of persistent viruses in the cell lines are too low to have an impact on RNAi efficiency in the lepidopteran cell lines and that encoded VSRs act locally at the sites of viral replication (mitochondrial membranes) without affecting the rest of the cytoplasm.

Specific Recognition of a Single-Stranded RNA Sequence by a Synthetic Antibody Fragment

Antibodies that bind RNA represent an unrealized source of reagents for synthetic biology and for characterizing cellular transcriptomes. However, facile access to RNA-binding antibodies requires the engineering of effective Fab libraries guided by the knowledge of the principles that govern RNA recognition. Here, we describe a Fab identified from a minimalist synthetic library during phage display against a branched RNA target. The Fab (BRG) binds with 20nM dissociation constant to a single-stranded RNA (ssRNA) sequence adjacent to the branch site and can block the action of debranchase enzyme. We report the crystal structure in complex with RNA target at 2.38Å. The Fab traps the RNA in a hairpin conformation that contains a 2-bp duplex capped by a tetraloop. The paratope surface consists of residues located in four complementarity-determining regions including a major contribution from H3, which adopts a helical structure that projects into a deep, wide groove formed by the RNA. The amino acid composition of the paratope reflects the library diversity, consisting mostly of tyrosine and serine residues and a small but significant contribution from a single arginine residue. This structure, involving the recognition of ssRNA via a stem–loop conformation, together with our two previous structures involving the recognition of an RNA hairpin loop and an RNA tertiary structure, reveals the capacity of minimalist libraries biased with tyrosine, serine, glycine, and arginine to form binding surfaces for specific RNA conformations and distinct levels of RNA structural hierarchy.

Advanced Design of Dumbbell-shaped Genetic Minimal Vectors Improves Non-coding and Coding RNA Expression

Dumbbell-shaped DNA minimal vectors lacking nontherapeutic genes and bacterial sequences are considered a stable, safe alternative to viral, nonviral, and naked plasmid-based gene-transfer systems. We investigated novel molecular features of dumbbell vectors aiming to reduce vector size and to improve the expression of noncoding or coding RNA. We minimized small hairpin RNA (shRNA) or microRNA (miRNA) expressing dumbbell vectors in size down to 130 bp generating the smallest genetic expression vectors reported. This was achieved by using a minimal H1 promoter with integrated transcriptional terminator transcribing the RNA hairpin structure around the dumbbell loop. Such vectors were generated with high conversion yields using a novel protocol. Minimized shRNA-expressing dumbbells showed accelerated kinetics of delivery and transcription leading to enhanced gene silencing in human tissue culture cells. In primary human T cells, minimized miRNA-expressing dumbbells revealed higher stability and triggered stronger target gene suppression as compared with plasmids and miRNA mimics. Dumbbell-driven gene expression was enhanced up to 56- or 160-fold by implementation of an intron and the SV40 enhancer compared with control dumbbells or plasmids. Advanced dumbbell vectors may represent one option to close the gap between durable expression that is achievable with integrating viral vectors and short-term effects triggered by naked RNA.

Magnetic tweezers force calibration for molecules that exhibit conformational switching.

High spatial and temporal resolution magnetic tweezers experiments allow for the direct calibration of pulling forces applied to short biomolecules. In one class of experiments, a force is applied to a structured RNA or protein to induce an unfolding transition; when the force is maintained at particular values, the molecule can exhibit conformational switching between the folded and unfolded states or between intermediate states. Here, we analyze the degree to which common force calibration approaches, involving the fitting of model functions to the Allan variance or power spectral density of the bead trajectory, are biased by this conformational switching. We find significant effects in two limits: that of large molecular extension changes between the two states, in which alternative fitting functions must be used, and that of very fast switching kinetics, in which the force calibration cannot be recovered due to the slow diffusion time of the magnetic bead. We use simulations and high-resolution RNA hairpin data to show that most biophysical experiments do not occur in either of these limits.

Silencing of TBX20 gene expression in rat myocardial and human embryonic kidney cells leads to cell cycle arrest in G2 phase.

Congenital heart diseases (CHDs) are the most common birth defects due to abnormal cardiac development. The T-box 20 (TBX20) gene is a member of the T-box family of transcription factors and encodes TBX20, which is essential for early heart development. In the present study, reduced TBX20 expression was observed in CHD tissue samples compared with normal tissues, and the function of TBX20 in Rattus norvegicus myocardial cells [H9c2(2-1)] and human embryonic kidney cells (HEK293) was investigated. TBX20 was silenced in H9c2 and HEK293 cells via transfection of small interfering RNA and short hairpin RNA duplexes, respectively, and TBX20 mRNA and protein levels were subsequently examined using reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and western blot analysis. Cell proliferation was assessed using a cell counting kit and proliferating cell nuclear antigen expression was determined by western blotting. Analysis of cell apoptosis was achieved by annexin V-fluorescein isothiocyanate/propidium iodide staining and a fluorometric terminal deoxynucleotidyl transferase dUTP nick-end labeling system. Cell cycle analysis was achieved using fluorescence-activated cell sorting, and, an RT-qPCR array was used to profile the expression of TBX20-related genes. Silencing of TBX20 in H9c2 and HEK293 cells significantly inhibited cell proliferation, induced cell apoptosis and led to G2/M cell cycle arrest. A reduction in cyclin B1 mRNA levels and an increase in cyclin-dependent kinase inhibitor 1B mRNA levels was observed, which indicated that cells were arrested in G2 phase. Concurrently, the mRNA levels of GATA binding protein 4 were increased in both cell lines, which may provide an explanation for the abnormal cardiac hypertrophy observed in patients with congenital heart disease. These results suggest that TBX20 is required for heart morphogenesis, and inhibition of TBX20 expression may lead to the suppression of cell proliferation and cell cycle arrest.

Effect of mutations on internal dynamics of an RNA hairpin from hepatitis B virus

Conformational dynamics plays a key role in mediating specific interactions between RNAs and proteins. Flexible parts, such as the loop regions, are often involved in protein binding. Characterization of the factors that influence the flexibility of loop regions will improve our understanding of RNA-protein binding. Here we use molecular dynamics simulations to study the dynamical features of the apical stem-loop of hepatitis B virus and a mutant, with two consensus-based secondary structure mutations (A-U→C-G) in the stem region. The mutations reduce the dynamics of the system and influence the hairpin conformations. The simulations show that inducing rigidity in the stem affects the loop conformational flexibility: the loop residues become less mobile and less accessible to the solvent, and thus less accessible to a possible targeting protein.

An Evolved RNA Recognition Motif That Suppresses HIV-1 Tat/TAR-Dependent Transcription.

Potent and selective recognition and modulation of disease-relevant RNAs remain a daunting challenge. We previously examined the utility of the U1A N-terminal RNA recognition motif as a scaffold for tailoring new RNA hairpin recognition and showed that as few as one or two mutations can result in moderate affinity (low μM dissociation constant) for the human immunodeficiency virus (HIV) trans-activation response element (TAR) RNA, an RNA hairpin controlling transcription of the human immunodeficiency virus (HIV) genome. Here, we use yeast display and saturation mutagenesis of established RNA-binding regions in U1A to identify new synthetic proteins that potently and selectively bind TAR RNA. Our best candidate has truly altered, not simply broadened, RNA-binding selectivity; it binds TAR with subnanomolar affinity (apparent dissociation constant of ∼0.5 nM) but does not appreciably bind the original U1A RNA target (U1hpII). It specifically recognizes the TAR RNA hairpin in the context of the HIV-1 5'-untranslated region, inhibits the interaction between TAR RNA and an HIV trans-activator of transcription (Tat)-derived peptide, and suppresses Tat/TAR-dependent transcription. Proteins described in this work are among the tightest TAR RNA-binding reagents-small molecule, nucleic acid, or protein-reported to date and thus have potential utility as therapeutics and basic research tools. Moreover, our findings demonstrate how a naturally occurring RNA recognition motif can be dramatically resurfaced through mutation, leading to potent and selective recognition-and modulation-of disease-relevant RNA.

JAR3D Webserver: Scoring and aligning RNA loop sequences to known 3D motifs.

Many non-coding RNAs have been identified and may function by forming 2D and 3D structures. RNA hairpin and internal loops are often represented as unstructured on secondary structure diagrams, but RNA 3D structures show that most such loops are structured by non-Watson–Crick basepairs and base stacking. Moreover, different RNA sequences can form the same RNA 3D motif. JAR3D finds possible 3D geometries for hairpin and internal loops by matching loop sequences to motif groups from the RNA 3D Motif Atlas, by exact sequence match when possible, and by probabilistic scoring and edit distance for novel sequences. The scoring gauges the ability of the sequences to form the same pattern of interactions observed in 3D structures of the motif. The JAR3D webserver at http://rna.bgsu.edu/jar3d/ takes one or many sequences of a single loop as input, or else one or many sequences of longer RNAs with multiple loops. Each sequence is scored against all current motif groups. The output shows the ten best-matching motif groups. Users can align input sequences to each of the motif groups found by JAR3D. JAR3D will be updated with every release of the RNA 3D Motif Atlas, and so its performance is expected to improve over time.

Nuclear factor erythroid-derived 2-like 2 (NFE2L2, Nrf2) mediates exercise-induced mitochondrial biogenesis and the anti-oxidant response in mice.

Reactive oxygen species (ROS) and nitric oxide (NO) regulate exercise-induced nuclear factor erythroid 2-related factor 2 (NFE2L2) expression in skeletal muscle. NFE2L2 is required for acute exercise-induced increases in skeletal muscle mitochondrial biogenesis genes, such as nuclear respiratory factor 1 (NRF-1) and mitochondrial transcription factor A, and anti-oxidant genes, such as superoxide dismutase (SOD)1, SOD2 and catalase. Following exercise training mice with impaired NFE2L2 expression have reduced exercise performance, energy expenditure, mitochondrial volume and anti-oxidant activity. In muscle cells, ROS and NO can regulate mitochondrial biogenesis via a NFE2L2/NRF-1-dependent pathway. Regular exercise induces adaptations to skeletal muscle, which can include mitochondrial biogenesis and enhanced anti-oxidant reserves. These adaptations and others are at least partly responsible for the improved health of physically active individuals. Reactive oxygen species (ROS) and nitric oxide (NO) are produced during exercise and may mediate the adaptive response to exercise in skeletal muscle. However, the mechanisms through which they act are unclear. In the present study, we aimed to determine the role of the redox-sensitive transcription factor nuclear factor erythroid-derived 2-like 2 (NFE2L2) in acute exercise- and training-induced mitochondrial biogenesis and the anti-oxidant response. We report that ROS and NO regulate acute exercise-induced expression of NFE2L2 in mouse skeletal muscle and muscle cells, and that deficiency in NFE2L2 prevents normal acute treadmill exercise-induced increases in mRNA of the mitochondrial biogenesis markers, nuclear respiratory factor 1 (NRF-1) and mitochondrial transcription factor A (mtTFA), and the anti-oxidants superoxide dismutase (SOD) 1 and 2, as well as catalase, in mouse gastrocnemius muscle. Furthermore, after 5weeks of treadmill exercise training, mice deficient in NFE2L2 had reduced exercise capacity and whole body energy expenditure, as well as skeletal muscle mitochondrial mass and SOD activity, compared to wild-type littermates. In C2C12 myoblasts, acute treatment with exogenous H2 O2 (ROS)- and diethylenetriamine/NO adduct (NO donor) induced increases in mtTFA, which was prevented by small interfering RNA and short hairpin RNA knockdown of either NFE2L2 or NRF-1. Our results suggest that, during exercise, ROS and NO can act via NFE2L2 to functionally regulate skeletal muscle mitochondrial biogenesis and anti-oxidant defence gene expression.

A combinatorial approach to the repertoire of RNA kissing motifs; towards multiplex detection by switching hairpin aptamers.

Loop–loop (also known as kissing) interactions between RNA hairpins are involved in several mechanisms in both prokaryotes and eukaryotes such as the regulation of the plasmid copy number or the dimerization of retroviral genomes. The stability of kissing complexes relies on loop parameters (base composition, sequence and size) and base combination at the loop–loop helix - stem junctions. In order to identify kissing partners that could be used as regulatory elements or building blocks of RNA scaffolds, we analysed a pool of 5.2 × 106 RNA hairpins with randomized loops. We identified more than 50 pairs of kissing RNA hairpins. Two kissing motifs, 5′CCNY and 5′RYRY, generate highly stable complexes with KDs in the low nanomolar range. Such motifs were introduced in the apical loop of hairpin aptamers that switch between unfolded and folded state upon binding to their cognate target molecule, hence their name aptaswitch. The aptaswitch–ligand complex is specifically recognized by a second RNA hairpin named aptakiss through loop–loop interaction. Taking advantage of our kissing motif repertoire we engineered aptaswitch–aptakiss modules for purine derivatives, namely adenosine, GTP and theophylline and demonstrated that these molecules can be specifically and simultaneously detected by surface plasmon resonance or by fluorescence anisotropy.

Direct-methods structure determination of a trypanosome RNA-editing substrate fragment with translational pseudosymmetry.

Using direct methods starting from random phases, the crystal structure of a 32-base-pair RNA (675 non-H RNA atoms in the asymmetric unit) was determined using only the native diffraction data (resolution limit 1.05 Å) and the computer program SIR2014. The almost three helical turns of the RNA in the asymmetric unit introduced partial or imperfect translational pseudosymmetry (TPS) that modulated the intensities when averaged by the l Miller indices but still escaped automated detection. Almost six times as many random phase sets had to be tested on average to reach a correct structure compared with a similar-sized RNA hairpin (27 nucleotides, 580 non-H RNA atoms) without TPS. More sensitive methods are needed for the automated detection of partial TPS.

A novel cytoprotective function for the DNA repair protein Ku in regulating p53 mRNA translation andfunction.

Ku heterodimer is a DNA binding protein with a prominent role in DNA repair. Here, we investigate whether and how Ku impacts the DNA damage response by acting as a post-transcriptional regulator of gene expression. We show that Ku represses p53 protein synthesis and p53-mediated apoptosis by binding to a bulged stem-loop structure within the p53 5' UTR However, Ku-mediated translational repression of the p53 mRNA is relieved after genotoxic stress. The underlying mechanism involves Ku acetylation which disrupts Ku-p53 mRNA interactions. These results suggest that Ku-mediated repression of p53 mRNA translation constitutes a novel mechanism linking DNA repair and mRNA translation.