RNA Hairpin Research Articles

Structural basis of Q-dependent transcription antitermination

Bacteriophage Q protein engages σ-dependent paused RNA polymerase (RNAP) by binding to a DNA site embedded in late gene promoter and renders RNAP resistant to termination signals. Here, we report a single-particle cryo-electron microscopy (cryo-EM) structure of an intact Q-engaged arrested complex. The structure reveals key interactions responsible for σ-dependent pause, Q engagement, and Q-mediated transcription antitermination. The structure shows that two Q protomers (QI and QII) bind to a direct-repeat DNA site and contact distinct elements of the RNA exit channel. Notably, QI forms a narrow ring inside the RNA exit channel and renders RNAP resistant to termination signals by prohibiting RNA hairpin formation in the RNA exit channel. Because the RNA exit channel is conserved among all multisubunit RNAPs, it is likely to serve as an important contact site for regulators that modify the elongation properties of RNAP in other organisms, as well.

Conjugation of N‐(3‐(9‐Ethynyl‐6H‐indolo[2,3‐b]quinoxalin‐6‐yl)propyl)‐2,2,2‐trifluoroacetamide Intercalator to a Triplex Forming Oligonucleotide, a Three‐Way Junction, and a G‐Quadruplex

A new intercalating nucleic acid monomer Z comprising an 3‐(9‐((4‐oxyphenyl)ethynyl)‐6H‐indolo[2,3‐b]quinoxalin‐6‐yl)propan‐1‐amine moiety was synthesized. When Z was inserted into triplex forming oligonucleotides, high thermal stability was observed for its corresponding Hoogsteen‐type triplexes. Three‐way junction (TWJ) was studied by targeting a DNA strand to the foot of a DNA or an RNA hairpin. When Z was inserted into the DNA strand, this resulted in the highest increase of thermal melting ever reported for a TWJ modified by insertion of an intercalator into the junction site. Experiments with mismatches confirmed formation of the TWJ. Improvements in stability of a G‐quadruplex were achieved by insertion of the monomer Z by replacement of one of the nucleotides in the TGT loop. The first steps in the synthesis of the monomer Z were condensation of 5‐iodoisatin with o‐phenylene diamine under reflux in acetic acid and subsequent alkylation reaction with 2‐(3‐bromopropyl)isoindoline‐1,3‐dione followed by suitable reduction led to 3‐(9‐Iodo‐6H‐indolo[2,3‐b]quinoxalin‐6‐yl)propan‐1‐amine which in turn was treated with ethyl trifluoroacetate with formation of 2,2,2‐trifluoro‐N‐(3‐(9‐iodo‐6H‐indolo[2,3‐b]quinoxalin‐6‐yl)propyl)acetamide. This compound was reacted by Sonogashira coupling conditions to give the diol which was converted into the DMT‐protected phosphoramidite, which in turn was used to incorporate the monomer Z into oligonucleotides.

The glucocorticoid receptor DNA-binding domain recognizes RNA hairpin structures with high affinity.

The glucocorticoid receptor (GR) binds the noncoding RNA Gas5 via its DNA-binding domain (DBD) with functional implications in pro-apoptosis signaling. Here, we report a comprehensive in vitro binding study where we have determined that GR-DBD is a robust structure-specific RNA-binding domain. GR-DBD binds to a diverse range of RNA hairpin motifs, both synthetic and biologically derived, with apparent mid-nanomolar affinity while discriminating against uniform dsRNA. As opposed to dimeric recognition of dsDNA, GR-DBD binds to RNA as a monomer and confers high affinity primarily through electrostatic contacts. GR-DBD adopts a discrete RNA-bound state, as assessed by NMR, distinct from both free and DNA-bound. NMR and alanine mutagenesis suggest a heightened involvement of the C-terminal α-helix of the GR-DBD in RNA-binding. RNA competes for binding with dsDNA and occurs in a similar affinity range as dimer binding to the canonical DNA element. Given the prevalence of RNA hairpins within the transcriptome, our findings strongly suggest that many RNAs have potential to impact GR biology.

Single-molecule insights into the temperature and pressure dependent conformational dynamics of nucleic acids in the presence of crowders and osmolytes

In this review we discuss results from temperature and pressure dependent single-molecule Förster resonance energy transfer (smFRET) studies on nucleic acids in the presence of macromolecular crowders and organic osmolytes. As representative examples, we have chosen fragments of both DNAs and RNAs, i.e., a synthetic DNA hairpin, a human telomeric G-quadruplex and the microROSE RNA hairpin. To mimic the effects of intracellular components, our studies include the macromolecular crowding agent Ficoll, a copolymer of sucrose and epichlorohydrin, and the organic osmolytes trimethylamine N-oxide, urea and glycine as well as natural occurring osmolyte mixtures from deep sea organisms. Furthermore, the impact of mutations in an RNA sequence on the conformational dynamics is examined. Different from proteins, the effects of the osmolytes and crowding agents seem to strongly dependent on the structure and chemical make-up of the nucleic acid.

Synthetic 5' UTRs Can Either Up- or Downregulate Expression upon RNA-Binding Protein Binding.

The construction of complex gene-regulatory networks requires both inhibitory and upregulatory modules. However, the vast majority of RNA-based regulatory "parts" are inhibitory. Using a synthetic biology approach combined with SHAPE-seq, we explored the regulatory effect of RNA-binding protein (RBP)-RNA interactions in bacterial 5' UTRs. Bypositioning a library of RNA hairpins upstream of a reporter gene and co-expressing them with the matching RBP, we observed a set of regulatory responses, including translational stimulation, translational repression, and cooperative behavior. Our combined approach revealed three distinct states invivo: in the absence of RBPs, the RNA molecules can be found in either a molten state that is amenable to translation or a structured phase that inhibits translation. In the presence of RBPs, the RNA molecules are in a semi-structured phase with partial translational capacity. Our work provides new insight into RBP-based regulation and a blueprint for designing complete gene-regulatory circuits at the post-transcriptional level.

Host-Induced Gene Silencing of MoAP1 Confers Broad-Spectrum Resistance to Magnaporthe oryzae.

Rice blast caused by Magnaporthe oryzae (M. oryzae) is a major threat to global rice production. In recent years, small interference RNAs (siRNAs) and host-induced gene silencing (HIGS) has been shown to be new strategies for the development of transgenic plants to control fungal diseases and proved a useful tool to study gene function in pathogens. We here tested whether in vitro feeding artificial siRNAs (asiRNAs) could compromise M. oryzae virulence and in vivo HIGS technique could improve rice blast resistance. Our data revealed that silencing of M. oryzae MoAP1 by feeding asiRNAs targeting MoAP1 (i.e., asiR1245, asiR1362, and asiR1115) resulted in inhibited fungal growth, abnormal spores, and decreased pathogenicity. Among the asiRNAs, asiR1115 was the most inhibitory toward the rice blast fungus. Conversely, the asiRNAs targeting three other genes (i.e., MoSSADH, MoACT, and MoSOM1) had no effect on fungal growth. Transgenic rice plants expressing RNA hairpins targeting MoAP1 exhibited improved resistance to 11 tested M. oryzae strains. Confocal microscopy also revealed profoundly restricted appressoria and mycelia in rice blast-infected transgenic rice plants. Our results demonstrate that in vitro asiRNA and in vivo HIGS were useful protection approaches that may be valuable to enhance rice blast resistance.

Development of the binding molecules for the RNA higher-order structures based on the guanine-recognition by the G-clamp

RNA higher-order structures play an important role for control of the gene expression, and the small molecules binding to these structures have potential to act as interfering agents in the RNA-mediated-pathway. In this study, we synthesized new RNA binding molecules based on the G-clamp structure and evaluated their binding properties using the model RNA. The monomeric G-clamp ligand exhibited a fluorescence quenching with RNA-binding. The dimeric G-clamp ligand showed a significant fluorescence OFF/ON response to the RNA hairpin structure containing the guanines, indicating a high affinity of the G-clamp dimer to two neighboring guanines located in the RNA hairpin loop.

Sequence- And Structure-Specific Probing of RNAs by Short Nucleobase-Modified dsRNA-Binding PNAs Incorporating a Fluorescent Light-up Uracil Analog.

RNAs are emerging as important biomarkers and therapeutic targets. The strategy of directly targeting double-stranded RNA (dsRNA) by triplex-formation is relatively underexplored mainly due to the weak binding at physiological conditions for the traditional triplex-forming oligonucleotides (TFOs). Compared to DNA and RNA, peptide nucleic acids (PNAs) are chemically stable and have a neutral peptide-like backbone, and thus, they show significantly enhanced binding to natural nucleic acids. We have successfully developed nucleobase-modified dsRNA-binding PNAs (dbPNAs) to facilitate structure-specific and selective recognition of dsRNA over single-stranded RNA (ssRNA) and dsDNA regions at near-physiological conditions. The triplex formation strategy facilitates the targeting of not only the sequence but also the secondary structure of RNA. Here, we report the development of novel dbPNA-based fluorescent light-up probes through the incorporation of A-U pair-recognizing 5-benzothiophene uracil (btU). The incorporation of btU into dbPNAs does not affect the binding affinity toward dsRNAs significantly, in most cases, as evidenced by our nondenaturing gel shift assay data. The blue fluorescence emission intensity of btU-modified dbPNAs is sequence- and structure-specifically enhanced by dsRNAs, including the influenza viral RNA panhandle duplex and HIV-1-1 ribosomal frameshift-inducing RNA hairpin, but not ssRNAs or DNAs, at 200 mM NaCl, pH 7.5. Thus, dbPNAs incorporating btU-modified and other further modified fluorescent nucleobases will be useful biochemical tools for probing and detecting RNA structures, interactions, and functions.

RhoTermPredict: an algorithm for predicting Rho-dependent transcription terminators based on Escherichia coli, Bacillus subtilis and Salmonella enterica databases.

BackgroundIn bacterial genomes, there are two mechanisms to terminate the DNA transcription: the “intrinsic” or Rho-independent termination and the Rho-dependent termination. Intrinsic terminators are characterized by a RNA hairpin followed by a run of 6–8 U residues relatively easy to identify using one of the numerous available prediction programs. In contrast, Rho-dependent termination is mediated by the Rho protein factor that, firstly, binds to ribosome-free mRNA in a site characterized by a C > G content and then reaches the RNA polymerase to induce its release. Conversely on intrinsic terminators, the computational prediction of Rho-dependent terminators in prokaryotes is a very difficult problem because the sequence features required for the function of Rho are complex and poorly defined. This is the reason why it still does not exist an exhaustive Rho-dependent terminators prediction program.ResultsIn this study we introduce RhoTermPredict, the first published algorithm for an exhaustive Rho-dependent terminators prediction in bacterial genomes. RhoTermPredict identifies these elements based on a previously proposed consensus motif common to all Rho-dependent transcription terminators. It essentially searches for a 78 nt long RUT site characterized by a C > G content and with regularly spaced C residues, followed by a putative pause site for the RNA polymerase. We tested RhoTermPredict performances by using available genomic and transcriptomic data of the microorganism Escherichia coli K-12, both in limited-length sequences and in the whole-genome, and available genomic sequences from Bacillus subtilis 168 and Salmonella enterica LT2 genomes. We also estimated the overlap between the predictions of RhoTermPredict and those obtained by the predictor of intrinsic terminators ARNold webtool. Our results demonstrated that RhoTermPredict is a very performing algorithm both for limited-length sequences (F1-score obtained about 0.7) and for a genome-wide analysis. Furthermore the degree of overlap with ARNold predictions was very low.ConclusionsOur analysis shows that RhoTermPredict is a powerful tool for Rho-dependent terminators search in the three analyzed genomes and could fill this gap in computational genomics. We conclude that RhoTermPredict could be used in combination with an intrinsic terminators predictor in order to predict all the transcription terminators in bacterial genomes.

A T7 autogene-based hybrid mRNA/DNA system for long-term shRNA expression in cytoplasm without inefficient nuclear entry

The transient silencing effects currently demonstrated by nonviral siRNA delivery systems limit the therapeutic utility of RNAi, but it remains a technical challenge to prolong duration of gene silencing. We have developed a T7 autogene-based hybrid mRNA/DNA system to enable long-term expression of shRNA in cytoplasm in vitro and in vivo. This hybrid mRNA/DNA system consists of T7 polymerase (T7pol) mRNA, pT7/shRNA-encoding DNA fragment and T7 autogene plasmid, and it can generate higher levels of T7pol proteins, compared to pCMV-triggering T7 autogene system, especially without the need of nuclear entry of any gene. A large amount of T7pol proteins produced are used to induce pT7-driven expression of shRNA in cytoplasm, and through cellular processing of RNA hairpins, mature siRNAs are generated for more than 13 days. We here demonstrate that a single liposomal delivery of this hybrid system leads to the long-term silencing effects in vitro and in vivo, in contrast to the conventional siRNA methods relying on the repeated administrations every 2 or 3 days. These sustainable shRNA expression properties in cytoplasm can provide an efficient strategy to address the limitations caused by shRNA-encoding plasmid DNA systems such as low nuclear entry efficiency and short-term silencing effect. The development of long-term shRNA expression system in vivo could scale down administration frequency of RNAi therapeutics in the treatment of chronic diseases, thereby increasing its clinical utility.

Metformin protects PC12 cells and hippocampal neurons from H2 O 2 -induced oxidative damage through activation of AMPK pathway.

Metformin, a first line anti type 2 diabetes drug, has recently been shown to extend lifespan in various species, and therefore, became the first antiaging drug in clinical trial. Oxidative stress due to excess reactive oxygen species (ROS) is considered to be an important factor in aging and related disease, such as Alzheimer's disease (AD). However, the antioxidative effects of metformin and its underlying mechanisms in neuronal cells is not known. In the present study, we showed that metformin, in clinically relevant concentrations, protected neuronal PC12 cells from H2 O2 -induced cell death. Metformin significantly ameliorated cell death due to H2 O2 insult by restoring abnormal changes in nuclear morphology, intracellular ROS, lactate dehydrogenase, and mitochondrial membrane potential induced by H2 O2 . Hoechst staining assay and flow cytometry analysis revealed that metformin significantly reduced the apoptosis in PC12 cells exposed to H2 O2 . Western blot analysis further demonstrated that metformin stimulated the phosphorylation and activation of AMP-activated protein kinase (AMPK) in PC12 cells, while application of AMPK inhibitor compound C, or knockdown of the expression of AMPK by specific small interfering RNA or short hairpin RNA blocked the protective effect of metformin. Similar results were obtained in primary cultured hippocampal neurons. Taken together, these results indicated that metformin is able to protect neuronal cells from oxidative injury, at least in part, via the activation of AMPK. As metformin is comparatively cheaper with much less side effects in clinic, our findings support its potential to be a drug for prevention and treatment of aging and aging-related diseases.

Structural Basis for the Action of an All-Purpose Transcription Anti-termination Factor

Bacteriophage λN protein, a model anti-termination factor, binds nascent RNA and host Nus factors, rendering RNA polymerase resistant to all pause and termination signals. A 3.7-Å-resolution cryo-electron microscopy structure and structure-informed functional analyses reveal a multi-pronged strategy by which the intrinsically unstructured λN directly modifies RNA polymerase interactions with the nucleic acids and subverts essential functions of NusA, NusE, and NusG to reprogram the transcriptional apparatus. λN repositions NusA and remodels the β subunit flap tip, which likely precludes folding of pause or termination RNA hairpins in the exit tunnel and disrupts termination-supporting interactions of the α subunit C-terminal domains. λN invades and traverses the RNA polymerase hybrid cavity, likely stabilizing the hybrid and impeding pause- or termination-related conformational changes of polymerase. λN also lines upstream DNA, seemingly reinforcing anti-backtracking and anti-swiveling by NusG. Moreover, λN-repositioned NusA and NusE sequester the NusG C-terminal domain, counteracting ρ-dependent termination. Other anti-terminators likely utilize similar mechanisms to enable processive transcription.

Modulating Responses of Toehold Switches by an Inhibitory Hairpin.

The toehold switch consists of a cis-repressing switch RNA hairpin and a trans-acting trigger RNA. The binding of the trigger RNA to an unpaired toehold sequence of the switch hairpin allows for a branch migration process, exposing the start codon and ribosome binding site for translation initiation. In this work, we demonstrate that responses of toehold switches can be modulated by introducing an inhibitory hairpin that shortens the length of the unpaired toehold region. First, we investigated the effect of the toehold region length on output gene expression and showed that the second trigger RNA, which binds to the inhibitory hairpin, is necessary for output gene activation when the hairpin-to-hairpin spacing is short. Second, the apparent Hill coefficient was found generally to increase with decreasing hairpin-to-hairpin spacing or increasing hairpin number. This work expands the utility of toehold switches by providing a new way to modulate their response.

Single Molecule Unfolding of RNA Hairpins

Single Molecule Unfolding of RNA Hairpins

Wnt7a predicts poor prognosis, and contributes to growth and metastasis in tongue squamous cell carcinoma.

Regional and distant metastases are the principal reasons underlying the high mortality rate associated with tongue squamous cell carcinoma (TSCC); however, the precise molecular mechanisms involved in tongue tumorigenesis remain unknown. The present study aimed to determine the expression and mechanism of regulation of Wnt7a in the growth and metastasis of TSCC. Wnt7a mRNA and protein expression levels were examined in TSCC tissues using reverse transcription‑quantitative polymerase chain reaction and immunohistochemical staining. A loss‑of‑function assay was performed in TSCC cell lines using Wnt7a small interfering RNA or short hairpin RNA, after which, cell proliferation, migration and invasion were analyzed using Cell Counting Kit‑8, tumorigenicity and Transwell assays, respectively. Epithelial‑mesenchymal transition (EMT)‑associated proteins were detected by western blotting. The mRNA and protein expression levels of Wnt7a were significantly upregulated in cancer tissues compared with in the adjacent non‑cancerous tissues. Clinical analysis indicated that Wnt7a expression was associated with T classification, lymph node metastasis and pathological differentiation, and high Wnt7a expression predicted a short recurrence‑free survival for patients with TSCC. Silencing Wnt7a expression suppressed cell proliferation, migration and invasion, and reversed the EMT phenotype in TSCC cell lines. The present study revealed that Wnt7a may be upregulated in TSCC, where it may participate in modulating cell proliferation, migration, invasion and the EMT of TSCC. Therefore, Wnt7a should be considered a novel oncogene, and a potential prognostic and therapeutic target for patients with TSCC.

The elemental mechanism of transcriptional pausing.

Transcriptional pausing underlies regulation of cellular RNA biogenesis. A consensus pause sequence that acts on RNA polymerases (RNAPs) from bacteria to mammals halts RNAP in an elemental paused state from which longer-lived pauses can arise. Although the structural foundations of pauses prolonged by backtracking or nascent RNA hairpins are recognized, the fundamental mechanism of the elemental pause is less well-defined. Here we report a mechanistic dissection that establishes the elemental pause signal (i) is multipartite; (ii) causes a modest conformational shift that puts γ-proteobacterial RNAP in an off-pathway state in which template base loading but not RNA translocation is inhibited; and (iii) allows RNAP to enter pretranslocated and one-base-pair backtracked states easily even though the half-translocated state observed in paused cryo-EM structures rate-limits pause escape. Our findings provide a mechanistic basis for the elemental pause and a framework to understand how pausing is modulated by sequence, cellular conditions, and regulators.

MRNA-Mediated Duplexes Play Dual Roles in the Regulation of Bidirectional Ribosomal Frameshifting.

In contrast to −1 programmed ribosomal frameshifting (PRF) stimulation by an RNA pseudoknot downstream of frameshifting sites, a refolding upstream RNA hairpin juxtaposing the frameshifting sites attenuates −1 PRF in human cells and stimulates +1 frameshifting in yeast. This eukaryotic functional mimicry of the internal Shine-Dalgarno (SD) sequence-mediated duplex was confirmed directly in the 70S translation system, indicating that both frameshifting regulation activities of upstream hairpin are conserved between 70S and 80S ribosomes. Unexpectedly, a downstream pseudoknot also possessed two opposing hungry codon-mediated frameshifting regulation activities: attenuation of +1 frameshifting and stimulation of a non-canonical −1 frameshifting within the +1 frameshift-prone CUUUGA frameshifting site in the absence of release factor 2 (RF2) in vitro. However, the −1 frameshifting activity of the downstream pseudoknot is not coupled with its +1 frameshifting attenuation ability. Similarly, the +1 frameshifting activity of the upstream hairpin is not required for its −1 frameshifting attenuation function Thus, each of the mRNA duplexes flanking the two ends of a ribosomal mRNA-binding channel possesses two functions in bi-directional ribosomal frameshifting regulation: frameshifting stimulation and counteracting the frameshifting activity of each other.

The DEK Oncoprotein Functions in Ovarian Cancer Growth and Survival

DNA damage repair alterations play a critical role in ovarian cancer tumorigenesis. Mechanistic drivers of the DNA damage response consequently present opportunities for therapeutic targeting. The chromatin-binding DEK oncoprotein functions in DNA double-strand break repair. We therefore sought to determine the role of DEK in epithelial ovarian cancer. DEK is overexpressed in both primary epithelial ovarian cancers and ovarian cancer cell lines. To assess the impact of DEK expression levels on cell growth, small interfering RNA and short hairpin RNA approaches were utilized. Decreasing DEK expression in ovarian cancer cell lines slows cell growth and induces apoptosis and DNA damage. The biologic effects of DEK depletion are enhanced with concurrent chemotherapy treatment. The in vitro effects of DEK knockdown are reproduced in vivo, as DEK depletion in a mouse xenograft model results in slower tumor growth and smaller tumors compared to tumors expressing DEK. These findings provide a compelling rationale to target the DEK oncoprotein and its pathways as a therapeutic strategy for treating epithelial ovarian cancer.

RNA accessibility impacts potency of Tough Decoy microRNA inhibitors

ABSTRACTMicroRNAs (miRNAs) are small RNA molecules that post-transcriptionally regulate gene expression through silencing of complementary target mRNAs. miRNAs are involved in many biological processes, including cell proliferation, differentiation, cell signaling and cellular defense responses to infection. Strategies that allow for strong and stable suppression of specific microRNA activity are needed to study miRNA functions and to develop therapeutic intervention strategies aimed at interfering with miRNA activity in vivo. One of these classes of miRNA inhibitors are Tough Decoys (TuD) RNAs, which comprise of an imperfect RNA hairpin structure that harbors two opposing miRNA binding sites. Upon developing TuDs targeting Epstein-Barr virus miRNAs, we observed a strong variation in inhibitory potential between different TuD RNAs targeting the same miRNA. We show that the composition of the ‘bulge’ sequence in the miRNA binding sites has a strong impact on the inhibitory potency of the TuD. Our data implies that miRNA inhibition correlates with the thermodynamic properties of the TuD and that design aimed at lowering the TuD opening energy increases TuD potency. Our study provides specific guidelines for the design and construction of potent decoy-based miRNA inhibitors, which may be used for future therapeutic intervention strategies.

Chemically Accurate Relative Folding Stability of RNA Hairpins from Molecular Simulations.

To benchmark RNA force fields, we compared the folding stabilities of three 12-nucleotide hairpin stem loops estimated by simulation to stabilities determined by experiment. We used umbrella sampling and a reaction coordinate of end-to-end (5' to 3' hydroxyl oxygen) distance to estimate the free energy change of the transition from the native conformation to a fully extended conformation with no hydrogen bonds between non-neighboring bases. Each simulation was performed four times using the AMBER FF99+bsc0+χOL3 force field, and each window, spaced at 1 Å intervals, was sampled for 1 μs, for a total of 552 μs of simulation. We compared differences in the simulated free energy changes to analogous differences in free energies from optical melting experiments using thermodynamic cycles where the free energy change between stretched and random coil sequences is assumed to be sequence-independent. The differences between experimental and simulated ΔΔ G° are, on average, 0.98 ± 0.66 kcal/mol, which is chemically accurate and suggests that analogous simulations could be used predictively. We also report a novel method to identify where replica free energies diverge along a reaction coordinate, thus indicating where additional sampling would most improve convergence. We conclude by discussing methods to more economically perform these simulations.