RNA Hairpin Research Articles

Improved microRNA suppression by WPRE-linked tough decoy microRNA sponges.

Our genes are post-transcriptionally regulated by microRNAs (miRNAs) inducing translational suppression and degradation of targeted mRNAs. Strategies to inhibit miRNAs in a spatiotemporal manner in a desired cell type or tissue, or at a desired developmental stage, can be crucial for understanding miRNA function and for pushing forward miRNA suppression as a feasible rationale for genetic treatment of disease. For such purposes, RNA polymerase II (RNA Pol II)-transcribed tough decoy (TuD) miRNA inhibitors are particularly attractive. Here, we demonstrate augmented miRNA suppression capacity of TuD RNA hairpins linked to the Woodchuck hepatitis virus post-transcriptional regulatory element (WPRE). This effect is position-dependent and evident only when the WPRE is positioned upstream of the TuD. In accordance, inclusion of the WPRE does not change nuclear export, translation, total levels of TuD-containing RNA transcripts, or cytoplasmic P-body localization, suggesting that previously reported WPRE functions are negligible for improved TuD function. Notably, deletion analysis of TuD-fused WPRE unveils truncated WPRE variants resulting in optimized miRNA suppression. Together, our findings add to the guidelines for production of WPRE-supported anti-miRNA TuDs.

Water isotope effect on the thermostability of a polio viral RNA hairpin: A metadynamics study.

Oral polio vaccine is considered to be the most thermolabile of all the common childhood vaccines. Despite heavy water (D2O) having been known for a long time to stabilise attenuated viral RNA against thermodegradation, the molecular underpinnings of its mechanism of action are still lacking. Whereas, understanding the basis of D2O action is an important step that might reform the way other thermolabile drugs are stored and could possibly minimize the cold chain problem. Here using a combination of parallel tempering and well-tempered metadynamics simulation in light water (H2O) and in D2O, we have fully described the free energy surface associated with the folding/unfolding of a RNA hairpin containing a non-canonical basepair motif, which is conserved within the 3'-untranslated region of poliovirus-like enteroviruses. Simulations reveal that in heavy water (D2O) there is a considerable increase of the stability of the folded basin as monitored through an intramolecular hydrogen bond (HB), size, shape, and flexibility of RNA structures. This translates into a higher melting temperature in D2O by 41 K when compared with light water (H2O). We have explored the hydration dynamics of the RNA, hydration shell around the RNA surface, and spatial dependence of RNA-solvent collective HB dynamics in the two water systems. Simulation in heavy water clearly showed that D2O strengthens the HB network in the solvent, lengthens inter-residue water-bridge lifetime, and weakens dynamical coupling of the hairpin to its solvation environment, which enhances the rigidity of solvent exposed sites of the native configurations. The results might suggest that like other added osmoprotectants, D2O can act as a thermostabilizer when used as a solvent.

19 F NMR Spectroscopic Analysis of the Binding Modes in Triple-Helical Peptide Nucleic Acid (PNA)/MicroRNA Complexes.

Triplex-forming peptide nucleic acids (TFPNAs) were targeted to double-helical regions of 19 F-labeled RNA hairpin models (a UA-rich duplex with a hexaethylene glycol (heg) loop and a microRNA model, miR-215). In addition to conventional UV- and circular dichroism (CD)-based detection, binding was monitored by 19 F NMR spectroscopy. Detailed information on the stoichiometry and transition between the triple-helical peptide nucleic acid (PNA)/RNA and (PNA)2 /RNA binding modes could be obtained. γ-(R)-Hydroxymethyl-modified thymine-1-yl- and 2-aminopyridin-3-yl-acetyl derivatives of TFPNAs were additionally synthesized, which were targeted to the same RNA models, and the effect of the γ-(R)-hydroxymethyl group on binding was studied. An appropriate pattern of γ-(R)-hydroxymethyl modifications reduced the stability of the ternary complex and preferred stoichiometric binding to the miR-215 model.

Improving Computational Predictions of Single-Stranded RNA Tetramers with Revised α/γ Torsional Parameters for the Amber Force Field.

With current advancements in RNA based therapeutics, it is becoming crucial to utilize theoretical and computational methods to describe properly the physical properties of RNA molecules. NMR and X-ray crystallography are two powerful techniques for investigating structural properties. However, if the RNA molecules are complex or dynamic, these methods might not be adequate. For computational approaches, the quality of the force field will determine accuracy of our predictions. In this contribution, we revise the α/γ torsional parameters of RNA for amber force field using a model system representing an RNA dimer backbone. Combined with revised χ torsional parameters, previously shown to improve computational predictions, we benchmarked the revised force field on five single-stranded RNA (ssRNA) tetramers, three RNA dodecamer duplexes, and an RNA hairpin. A total of 60 μs of molecular dynamics (MD) simulations were run. We also employ the discrete path sampling (DPS) approach to compare the predictions for the revised amber force field with those for amber10. Our results indicate that the unphysical states observed with amber10 in ssRNA MD simulations are suppressed for the revised amber force field. In line with NMR experimental observations, incorporation of the revised α/γ and χ torsional parameters leads to A-form-like conformational states as the most favorable ssRNA tetramer conformations. Furthermore, the revised force field maintains the A-form geometry in regular RNA duplexes. Our revised amber force field for RNA should therefore improve structural and thermodynamic predictions for challenging RNA systems.

Friend leukemia virus integration 1 promotes tumorigenesis of small cell lung cancer cells by activating the miR-17-92 pathway.

Small cell lung cancer (SCLC) is regarded as the most devastative type of human lung malignancies. The rapid and disseminated growth pattern remains the primary cause of poor clinical prognosis in patients with SCLC. However, the molecular factors that drive rapid progression of SCLC remain unclear. Friend leukemia virus integration 1 (FLI1), an Ets transcription factor family member, has been previously reported to act as a major driver of hematological malignancies. In this study, we explored the potential role of FLI1 in SCLC. Using immunohistochemical staining, we found that FLI1 was significantly upregulated in SCLC tissues, compared to that in non-small cell lung cancer (NSCLC) and normal lung tissues (p < 0.01). The expression score of FLI1 oncoprotein was associated with the extensive stage of SCLC and the overexpressed Ki67. Knockdown of FLI1 with small interfering RNA (siRNA) or short hairpin RNA (shRNA) promoted apoptosis and induced repression of cell proliferation, tumor colony formation and in vivo tumorigenicity in highly aggressive SCLC cell lines. Importantly, we discovered that FLI1 promoted tumorigenesis by activating the miR-17-92 cluster family. This study uncovers FLI1 as an important driving factor that promotes tumor growth in SCLC through the miR-17-92 pathway. FLI1 may serve as an attractive target for therapeutic intervention of SCLC.

Cytosolic YB-1 and NSUN2 are the only proteins recognizing specific motifs present in mRNAs enriched in exosomes

Exosomes, membranous vesicles secreted by various cells, are involved in intercellular communication and carry vast repertoires of RNAs and proteins. Processes mediating RNA sorting into exosomes are currently poorly understood. Using bioinformatics approaches, three structural motifs ACCAGCCU, CAGUGAGC and UAAUCCCA have been discovered as enriched in exosomal mRNAs and long noncoding RNAs. Here, utilizing short RNA hairpins, each containing one of the motifs, in a pull-down assay of cytosolic extract of human embryonic kidney 293 (HEK293) cells, we prove that multifunctional RNA-binding protein YB-1 specifically interacts with all three motifs, whereas methyltransferase NSUN2 recognizes only the motif CAGUGAGC. RNA hairpins other than those mentioned above pull out neither YB-1 nor NSUN2. Both these proteins are found in exosomes secreted by HEK293 cells. YB-1 for all that is detected as a form having a slightly higher electrophoretic mobility than that of YB-1 associated with the above RNA hairpins, assuming changes in posttranslational modifications of the protein during its transfer from cytoplasm into exosomes. Next generation sequencing of total exosomal RNA (eRNA) reveals a large representative set of RNA species, including mRNAs containing the above-mentioned motifs. The degree of enrichment in exosomes with this kind of mRNAs strongly depends on the locations of eRNA-specific motifs within the mRNA sequences. Altogether, our findings point to YB-1 and NSUN2 as possible mediators of the process of transfer of specific mRNAs into exosomes, allowing us to speculate on an involvement of these proteins in the mRNA sorting via the recognition of the above motifs.

Interleukin-17 augments tumor necrosis factor α-mediated increase of hypoxia-inducible factor-1α and inhibits vasodilator-stimulated phosphoprotein expression to reduce the adhesion of breast cancer cells.

Interleukin-17 (IL-17) and tumor necrosis factor (TNF)-α are able to cooperatively alter the expression levels of a number of genes. In the present study, the mRNA expression levels of hypoxia-inducible factor (HIF)-1α were analyzed in MDA-MB-231 breast cancer cells following treatment with IL-17, TNF-α or the combination of IL-17 and TNF-α. The protein expression levels of HIF-1α and vasodilator-stimulated phosphoprotein (VASP) were evaluated using western blot analysis. The adhesive ability of the cells was determined using an MTT assay following treatment with HIF-1α-small interfering RNA and short hairpin RNA-VASP that were used to suppress the expression levels of HIF-1α and VASP protein, respectively. These results demonstrated that IL-17 augmented TNF-α-induced gene expression of HIF-1α. The combination of IL-17 and TNF-α promoted an increase in HIF-1α expression and a decrease in VASP expression and a reduction in the adhesive ability of cells. These results demonstrated that IL-17 effectively enhanced the TNF-α-induced increase in HIF-1α and inhibited VASP expression, thus reducing the adhesion of MDA-MB-231 cells.

Direct Observation of Folding Energy Landscape of RNA Hairpin at Mechanical Loading Rates.

By applying a controlled mechanical load using optical tweezers, we measured the diffusive barrier crossing in a 49 nt long P5ab RNA hairpin. We find that in the free-energy landscape the barrier height (G‡) and transition distance (x‡) are dependent on the loading rate (r) along the pulling direction, x, as predicted by Bell. The barrier shifted toward the initial state, whereas ΔG‡ reduced significantly from 50 to 5 kT, as r increased from 0 to 32 pN/s. However, the equilibrium work (ΔG) during strand separation, as estimated by Crook's fluctuation theorem, remained unchanged at different rates. Previously, helix formation and denaturation have been described as two-state (F ↔ U) transitions for P5ab. Herein, we report three intermediate states I1, I, and I2 located at 4, 11, and 16 nm respectively, from the folded conformation. The intermediates were observed only when the hairpin was subjected to an optimal r, 7.6 pN/s. The results indicate that the complementary strands in P5ab can zip and unzip through complex routes, whereby mismatches act as checkpoints and often impose barriers. The study highlights the significance of loading rates in force-spectroscopy experiments that are increasingly being used to measure the folding properties of biomolecules.

Competitive folding of RNA structures at a termination-antitermination site.

Antitermination is a regulatory process based on the competitive folding of terminator–antiterminator structures that can form in the leader region of nascent transcripts. In the case of the Bacillus subtilis licS gene involved in β-glucosides utilization, the binding of the antitermination protein LicT to a short RNA hairpin (RAT) prevents the formation of an overlapping terminator and thereby allows transcription to proceed. Here, we monitored in vitro the competition between termination and antitermination by combining bulk and single-molecule fluorescence-based assays using labeled RNA oligonucleotide constructs of increasing length that mimic the progressive transcription of the terminator invading the antiterminator hairpin. Although high affinity binding is abolished as soon as the antiterminator basal stem is disrupted by the invading terminator, LicT can still bind and promote closing of the partially unfolded RAT hairpin. However, binding no longer occurs once the antiterminator structure has been disrupted by the full-length terminator. Based on these findings, we propose a kinetic competition model for the sequential events taking place at the termination–antitermination site, where LicT needs to capture its RAT target before completion of the terminator to remain tightly bound during RNAP pausing, before finally dissociating irreversibly from the elongated licS transcript.

Stabilization of RNA hairpins using non-nucleotide linkers and circularization.

An RNA hairpin is an essential structural element of RNA. Hairpins play crucial roles in gene expression and intermolecular recognition but are also involved in the pathogenesis of some congenital diseases. Structural studies of the hairpin motifs are impeded by their thermodynamic instability, as they tend to unfold to form duplexes, especially at high concentrations required for crystallography or nuclear magnetic resonance spectroscopy. We have elaborated techniques to stabilize the RNA hairpins by linking the free ends of the RNA strand at the base of the hairpin stem. One method involves stilbene diether or hexaethylene glycol linkers and circularization by T4 RNA ligase. Another method uses click chemistry to stitch the RNA ends with a triazole linker. Both techniques are efficient and easy to perform. They should be useful in making stable, biologically relevant RNA constructs for structural studies.

Equilibrium Denaturation and Preferential Interactions of an RNA Tetraloop with Urea

Urea is an important organic cosolute with implications in maintaining osmotic stress in cells and differentially stabilizing ensembles of folded biomolecules. We report an equilibrium study of urea-induced denaturation of a hyperstable RNA tetraloop through unbiased replica exchange molecular dynamics. We find that, in addition to destabilizing the folded state, urea smooths the RNA free energy landscape by destabilizing specific configurations, and forming favorable interactions with RNA nucleobases. A linear concentration-dependence of the free energy (m-value) is observed, in agreement with the results of other RNA hairpins and proteins. Additionally, analysis of the hydrogen-bonding and stacking interactions within RNA primarily show temperature-dependence, while interactions between RNA and urea primarily show concentration-dependence. Our findings provide valuable insight into the effects of urea on RNA folding and describe the thermodynamics of a basic RNA hairpin as a function of solution chemistry.

ERCC6L, a DNA helicase, is involved in cell proliferation and associated with survival and progress in breast and kidney cancers.

By analyzing 4987 cancer transcriptomes from The Cancer Genome Atlas (TCGA), we identified that excision repair cross-complementation group 6 like (ERCC6L), a newly discovered DNA helicase, is highly expressed in 12 solid cancers. However, its role and mechanism in tumorigenesis are largely unknown. In this study, we found that ERCC6L silencing by small interring RNA (siRNA) or short hairpin RNA (shRNA) significantly inhibited the proliferation of breast (MCF-7, MDA-MB-231) and kidney cancer cells (786-0). Furthermore, ERCC6L silencing induced cell cycle arrest at G0/G1 phase without affecting apoptosis. We then performed RNA sequencing (RNA-seq) analysis after ERCC6L silencing and identified that RAB31 was markedly downregulated at both the transcriptional and translational levels. Its downstream protein, phosphorylated MAPK and CDK2 were also inhibited by ERCC6L silencing. The xenograft experiment showed that silencing of ERCC6L strikingly inhibited tumor growth from the 7th day after xenograft in nude mice. In addition, higher ERCC6L expression was found to be significantly associated with worse clinical survival in breast and kidney cancers. In conclusion, our results suggest that ERCC6L may stimulates cancer cell proliferation by promoting cell cycle through a way of RAB31-MAPK-CDK2, and it could be a potential biomarker for cancer prognosis and target for cancer treatment.

Mechanical Characterization of the HIV-1 RNA Hairpin using an Atomic Force Microscope

Pioneering single-molecule force spectroscopy (SMFS) studies of RNA structures used a custom-built optical trap, which continues to be the preferred measurement platform due to its exquisite stability and force precision. Here, we developed a rapid and precise SMFS assay to characterize the dynamics and energetics of RNA structures using a commercial atomic force microscope (AFM) equipped with a temperature-regulated closed-fluidic sample chamber. To do so, we integrated several technical improvements: (i) focused-ion beam modified cantilevers to provide sub-pN stability in conjunction with fast temporal response (∼40 µs); (ii) site-specific attachment to covalently couple RNA hairpins to PEG-coated coverslips and to reversibly attach such hairpins to PEG-coated AFM tips via a streptavidin-biotin bond; and (iii) an automated centering routine to achieve a vertical pulling geometry. By leveraging these improvements, we resolved the unfolding and refolding of single RNA hairpins in both dynamic and equilibrium assays, including repeatedly measuring the same individual molecule over >1 h. Initial studies focused on the HIV-1 RNA hairpin, which leads to a −1 programmed ribosomal frameshift. Dynamic force spectroscopy analysis of the HIV-1 hairpin yielded ΔG‡ (= 36 kBT), Δx‡ (= 7.1 nm), and k0 (= 1.3×10−9 s−1). Looking forward, the rapid data acquisition demonstrated here on a commercial AFM facilitates SMFS studies of macromolecular folding over a broader a range of physiochemical conditions (pH, temperature, denaturant).

Precise quantification of translation inhibition by mRNA structures that overlap with the ribosomal footprint in N-terminal coding sequences.

A mRNA's translation rate is controlled by several sequence determinants, including the presence of RNA structures within the N-terminal regions of its coding sequences. However, the physical rules that govern when such mRNA structures will inhibit translation remain unclear. Here, we introduced systematically designed RNA hairpins into the N-terminal coding region of a reporter protein with steadily increasing distances from the start codon, followed by characterization of their mRNA and expression levels in Escherichia coli. We found that the mRNAs’ translation rates were repressed, by up to 530-fold, when mRNA structures overlapped with the ribosome's footprint. In contrast, when the mRNA structure was located outside the ribosome's footprint, translation was repressed by <2-fold. By combining our measurements with biophysical modeling, we determined that the ribosomal footprint extends 13 nucleotides into the N-terminal coding region and, when a mRNA structure overlaps or partially overlaps with the ribosomal footprint, the free energy to unfold only the overlapping structure controlled the extent of translation repression. Overall, our results provide precise quantification of the rules governing translation initiation at N-terminal coding regions, improving the predictive design of post-transcriptional regulatory elements that regulate translation rate.

Preferential Interactions of Charged and Neutral Cosolutes with a Model RNA Hairpin

Preferential Interactions of Charged and Neutral Cosolutes with a Model RNA Hairpin

RNA binding and chaperone activity of the E. coli cold-shock protein CspA.

Ensuring the correct folding of RNA molecules in the cell is of major importance for a large variety of biological functions. Therefore, chaperone proteins that assist RNA in adopting their functionally active states are abundant in all living organisms. An important feature of RNA chaperone proteins is that they do not require an external energy source to perform their activity, and that they interact transiently and non-specifically with their RNA targets. So far, little is known about the mechanistic details of the RNA chaperone activity of these proteins. Prominent examples of RNA chaperones are bacterial cold shock proteins (Csp) that have been reported to bind single-stranded RNA and DNA. Here, we have used advanced NMR spectroscopy techniques to investigate at atomic resolution the RNA-melting activity of CspA, the major cold shock protein of Escherichia coli, upon binding to different RNA hairpins. Real-time NMR provides detailed information on the folding kinetics and folding pathways. Finally, comparison of wild-type CspA with single-point mutants and small peptides yields insights into the complementary roles of aromatic and positively charged amino-acid side chains for the RNA chaperone activity of the protein.

Novel determinants of mammalian primary microRNA processing revealed by systematic evaluation of hairpin-containing transcripts and human genetic variation

Mature microRNAs (miRNAs) are processed from hairpin-containing primary miRNAs (pri-miRNAs). However, rules that distinguish pri-miRNAs from other hairpin-containing transcripts in the genome are incompletely understood. By developing a computational pipeline to systematically evaluate 30 structural and sequence features of mammalian RNA hairpins, we report several new rules that are preferentially utilized in miRNA hairpins and govern efficient pri-miRNA processing. We propose that a hairpin stem length of 36 ± 3 nt is optimal for pri-miRNA processing. We identify two bulge-depleted regions on the miRNA stem, located ∼16–21 nt and ∼28–32 nt from the base of the stem, that are less tolerant of unpaired bases. We further show that the CNNC primary sequence motif selectively enhances the processing of optimal-length hairpins. We predict that a small but significant fraction of human single-nucleotide polymorphisms (SNPs) alter pri-miRNA processing, and confirm several predictions experimentally including a disease-causing mutation. Our study enhances the rules governing mammalian pri-miRNA processing and suggests a diverse impact of human genetic variation on miRNA biogenesis.

Targeting deregulated AMPK/mTORC1 pathways improves muscle function in myotonic dystrophy type I.

Myotonic dystrophy type I (DM1) is a disabling multisystemic disease that predominantly affects skeletal muscle. It is caused by expanded CTG repeats in the 3'-UTR of the dystrophia myotonica protein kinase (DMPK) gene. RNA hairpins formed by elongated DMPK transcripts sequester RNA-binding proteins, leading to mis-splicing of numerous pre-mRNAs. Here, we have investigated whether DM1-associated muscle pathology is related to deregulation of central metabolic pathways, which may identify potential therapeutic targets for the disease. In a well-characterized mouse model for DM1 (HSALR mice), activation of AMPK signaling in muscle was impaired under starved conditions, while mTORC1 signaling remained active. In parallel, autophagic flux was perturbed in HSALR muscle and in cultured human DM1 myotubes. Pharmacological approaches targeting AMPK/mTORC1 signaling greatly ameliorated muscle function in HSALR mice. AICAR, an AMPK activator, led to a strong reduction of myotonia, which was accompanied by partial correction of misregulated alternative splicing. Rapamycin, an mTORC1 inhibitor, improved muscle relaxation and increased muscle force in HSALR mice without affecting splicing. These findings highlight the involvement of AMPK/mTORC1 deregulation in DM1 muscle pathophysiology and may open potential avenues for the treatment of this disease.

A colorimetric nanosensor based on a selective target-responsive aptamer kissing complex.

Herein, we report a novel approach for the design of a colorimetric aptasensor based on functionalized gold nanoparticle probes. This approach relies on the conjugation of nanoparticles by two functional DNA and RNA hairpins that engage specific kissing (loop-loop) interactions in response to the addition of a small analyte ligand, leading to particle aggregation and then red-to-purple colour change of the colloidal solution.

Origins of the enhanced affinity of RNA-protein interactions triggered by RNA phosphorodithioate backbone modification.

The well-characterized interaction between the MS2 coat protein and its cognate RNA hairpin was used to evaluate changes in affinity as a result of phosphorodithioate (PS2) replacing phosphate by biolayer interferometry (BLI). A structure-based analysis of the data provides insights into the origins of the enhanced affinity of RNA-protein interactions triggered by the PS2 moiety.