Short Stem-loop Structure Research Articles

M6A Methylation of Transcription Leader Sequence of SARS-CoV-2 Impacts Discontinuous Transcription of Subgenomic mRNAs.

The SARS-CoV-2 genome has been shown to be m6A methylated at several positions in vivo. Strikingly, a DRACH motif, the recognition motif for adenosine methylation, resides in the core of the transcriptional regulatory leader sequence (TRS-L) at position A74, which is highly conserved and essential for viral discontinuous transcription. Methylation at position A74 correlates with viral pathogenicity. Discontinuous transcription produces a set of subgenomic mRNAs that function as templates for translation of all structural and accessory proteins. A74 is base-paired in the short stem-loop structure 5'SL3 that opens during discontinuous transcription to form long-range RNA-RNA interactions with nascent (-)-strand transcripts at complementary TRS-body sequences. A74 can be methylated by the human METTL3/METTL14 complex in vitro. Here, we investigate its impact on the structural stability of 5'SL3 and the long-range TRS-leader:TRS-body duplex formation necessary for synthesis of subgenomic mRNAs of all four viral structural proteins. Methylation uniformly destabilizes 5'SL3 and long-range duplexes and alters their relative equilibrium populations, suggesting that the m6A74 modification acts as a regulator for the abundance of viral structural proteins due to this destabilization.

An ultrasensitive J-shaped optical fiber LSPR aptasensor for the detection of Helicobacter pylori

The development of label-free and sensitive detection of pathogenic bacteria is of great significance for disease prevention and public health protection. In this study, an originally bent structure, named as J-shaped optical fiber probe, was first designed to engineer a localized surface plasmon resonance (LSPR) aptamer biosensor for the rapid and ultrasensitive detection of Helicobacter pylori (H. pylori). The J-shaped optical fiber probe exhibited a significant improvement in refractive index sensitivity (RIS) and LSPR signal response. Meantime, the original sequence of aptamer was truncated in order to effectively capture H. pylori on the optical fiber surface. Besides, a spacer nucleic acid with short stem-loop structure was adopted to control the aptamer density on gold nanoparticles (AuNPs) on the surface of the J-shaped optical fiber probe, which displayed a further enhancement in LSPR signal response. Benefitting from these creative designs, the proposed LSPR biosensor can realize label-free and sensitive detection of H. pylori with a detection limit as low as 45 CFU/mL and a wide linear range from 1.0 × 102 CFU/mL to 1.0 × 108 CFU/mL. At the same time, the sensing strategy can detect the pathogenic bacteria from actual water samples in one step just in 30 min without any sample pretreatment. Due to the advantages of ease-to-preparation, high sensitivity, and rapid analysis, this proposed J-shaped optical fiber LSPR aptasensor can provide a potential strategy for point-of-caring detection of pathogenic bacteria in environmental monitoring and disease diagnosis.

Identification of a short form of a Caenorhabditis elegans Y RNA homolog Cel7 RNA

Cel7 RNA is a member of the Caenorhabditis elegans stem-bulge RNAs (sbRNAs) that are classified into the Y RNA family based on their structural similarity. We identified a 15-nucleotide-shorter form of Cel7 RNA and designated it Cel7s RNA. Both Cel7 and Cel7s RNAs increased during the development of worms from L1 to adult. Cel7s RNA was notably more abundant in embryos than in L1 to L3 larvae. Cel7 RNA in embryo was less than those in L2 to adult. The ratio of cellular level of Cel7 RNA to that of Cel7s RNA was higher in L1 to L4, but reversed in embryos and adults. In rop-1 mutants, in which the gene for the C. elegans Ro60 homolog, ROP-1, was disrupted, Cel7s RNA decreased similar to CeY RNA, another C. elegans Y RNA homolog. Surprisingly, Cel7 RNA, existed stably in the absence of ROP-1, unlike Cel7s and CeY RNAs. Gel-shift assays demonstrated that Cel7 and Cel7s RNAs bound to ROP-1 in a similar manner, which was much weaker than CeY RNA. The 5′-terminal 15-nt of Cel7 RNA could be folded into a short stem-loop structure, probably contributing to the stability of Cel7 RNA in vivo and the distinct expression patterns of the 2 RNAs.

Low Temperature Greatly Enhancing Responses of Aptamer Electrochemical Sensor for Aflatoxin B1 Using Aptamer with Short Stem.

Aflatoxin B1 (AFB1), one of the most toxic mycotoxins, poses great health risks. Rapid and sensitive detection of AFB1 is important for food safety, environment monitoring, and health risk assessment. We report here the development of a simple and reusable electrochemical aptasensor for rapid and sensitive detection of AFB1. Main improvements were achieved through engineering an aptamer containing a short stem-loop structure and enhancing the binding affinity at a lower temperature. The DNA aptamer with a methylene blue (MB) label at one end was immobilized on a gold electrode. Upon AFB1 binding, the aptamer folded into a stem-loop structure and brought MB close to the electrode surface, resulting in increases in electric current. The aptamer having a shorter stem (2-4 bp) underwent a larger conformation change upon target binding. The sensors built with the aptamer containing a 2 bp stem generated much higher signal-on responses to AFB1 at 4 °C than at room temperature (25 °C). The improvements resulted in a detection limit of 6 pM, enabling the determination of trace AFB1 in a complex sample matrix. This study demonstrates that low temperature greatly enhances the performance of aptamer electrochemical sensors. This aptasensor is simple to construct and readily regenerated by washing with deionized water for reuse. This aptasensor strategy could be applied to the development of an electrochemical aptasensor for other targets.

Looking for the key to secrets of RNA under the lamp-post.

Looking for the key to secrets of RNA under the lamp-post.

Abstract P4-07-19: MicroRNA miR-24 enhances tumor angiogenesis and metastasis in breast cancer

Abstract MicroRNAs (miRNA) are non-coding single-stranded RNA molecules, which are produced from endogenous transcripts of short stem-loop structures. Through binding to the 3’-untranslated region (3’UTR) of different target messenger RNAs (mRNAs), microRNAs can repress the translation of the target mRNAs. Recent studies have shown that miR-24 can inhibit apoptosis of cancer cells and cardiomyocytes, and can accelerate cell proliferation. Up-regulation of miR-24 has been observed in oral carcinoma and is one of the most abundant miRNAs in cervical cancer. However, the role of miR-24 in breast cancer cell angiogenesis and metastasis is not clear. We analyzed microRNA miR-24 levels in patients with breast carcinoma and found that miR-24 was higher in breast carcinoma samples than in breast benign tissues. We generated constructs expressing miR-24 and studied its functions using both in vitro and in vivo techniques. We found that the ectopic expression of miR-24 promoted breast cancer angiogenesis and metastasis. Cell migration assays indicated that expression of miR-24 promoted migration of MT-1 cells and 4T1 cells. We examined proliferation of MT-1 cells and 4T1 cells transfected with miR-24 or the control vector. The experiments showed that the miR-24 transfected cells had high rates of proliferation than the vector-transfected cells. In vivo experiments indicated that the expression of miR-24 enhanced tumor growth, metastasis to lung tissues, and decreased overall mouse survival. Tumor sections were also probed with anti-CD34 antibody to examine angiogenesis. We found that miR-24 expression increased tumor-associated vascularization. We further performed tumor metastatic assays. DNA was isolated from lung tissues and subjected to PCR to indicate metastasis of the cancer cells. Typical metastatic lesions in the lungs are shown. In the miR-24 expressing cells and tumors, expression of the phosphatases PTPN9 and PTPRF were repressed. We confirmed that miR-24 could directly target both PTPN9 and PTPRF. Consistent with this, we found that the levels of PTPN9 and PTPRF were lower in the patients with metastatic breast carcinoma. Ectopic expression of PTPN9 and PTPRF decreased cell migration, and tumor metastasis. Our study demonstrates that miR-24 is highly expressed in human breast carcinoma. This finding suggests a role of miR-24 in breast cancer development/progression. Our results suggest that miR-24 plays a key role in breast cancer angiogenesis and metastasis. miR-24 could potentially be a target for cancer intervention. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P4-07-19.

Exon Skipping of Hepatic APOB Pre-mRNA With Splice-switching Oligonucleotides Reduces LDL Cholesterol In Vivo

Familial hypercholesterolemia (FH) is a genetic disorder characterized by extremely high levels of plasma low-density lipoprotein (LDL), due to defective LDL receptor-apolipoprotein B (APOB) binding. Current therapies such as statins or LDL apheresis for homozygous FH are insufficiently efficacious at lowering LDL cholesterol or are expensive. Treatments that target APOB100, the structural protein of LDL particles, are potential therapies for FH. We have developed a series of APOB-directed splice-switching oligonucleotides (SSOs) that cause the expression of APOB87, a truncated isoform of APOB100. APOB87, like similarly truncated isoforms expressed in patients with a different condition, familial hypobetalipoproteinemia, lowers LDL cholesterol by inhibiting very low-density lipoprotein (VLDL) assembly and increasing LDL clearance. We demonstrate that these "APO-skip " SSOs induce high levels of exon skipping and expression of the APOB87 isoform, but do not substantially inhibit APOB48 expression in cell lines. A single injection of an optimized APO-skip SSO into mice transgenic for human APOB resulted in abundant exon skipping that persists for >6 days. Weekly treatments generated a sustained reduction in LDL cholesterol levels of 34-51% in these mice, superior to pravastatin in a head-to-head comparison. These results validate APO-skip SSOs as a candidate therapy for FH.

Coarse-Grained Model DNA with Explicit H-Bonding and Implicit Solvent

We present a coarse-grained model for DNA that is intended to function realistically at the level of individual bases. The model is composed of residues with up to eight coarse-grained beads each, which is sufficient for DNA-like base stacking and base-base recognition by hydrogen bonding. The beads interact by means of short-ranged pair potentials and a simple implicit solvent model. Movement is simulated by Brownian dynamics without hydrodynamic coupling. The main stabilizing forces are base stacking and hydrogen bonding, as modified by the effects of solvation. Complementary double-stranded chains of such residues form stable double helices over long runs (∼10 μs) at or near room temperature, with structural parameters close to those of B-form DNA. Most mismatched chains or mismatched regions within a complementary molecule melt and become disordered. Long-range fluctuations and elastic properties, as measured by bending and twisting persistence lengths, are close to experimental values. Single-stranded chains are flexible, with transient stretches of free bases in equilibrium with globules stabilized by intrastrand stacking and hydrogen bonding. Model DNAs in covalently closed loops form right or left-handed supercoils, depending on the sign of overtwist or undertwist. Short stem-loop structures melt at elevated temperatures and reanneal when the temperature is carefully lowered. Overall, most qualitative properties of real DNA arise naturally in the model from local interactions at the base pair level.

Coarse-Grained Model DNA: Structure, Sequences, Stems, Circles, Hairpins

A coarse-grained model for DNA that is intended to function realistically at the level of individual bases is reported. The model is composed of residues with up to eight coarse-grained beads each, which is sufficient for DNA-like base stacking and base-base recognition by hydrogen bonding. The beads interact by means of short-ranged pair potentials and a simple implicit solvent model. Movement is simulated by Brownian dynamics without hydrodynamic coupling. The main stabilizing forces are base stacking and hydrogen bonding, as modified by the effects of solvation. Complementary double-stranded chains of such residues form stable double helices over long runs (~10 μs) at or near room temperature, with structural parameters close to those of B-form DNA. Most mismatched chains or mismatched regions within a complementary molecule melt and become disordered. Long-range fluctuations and elastic properties, as measured by bending and twisting persistence lengths, are close to experimental values. Single-stranded chains are flexible, with transient stretches of free bases in equilibrium with globules stabilized by intrastrand stacking and hydrogen bonding. Model DNAs in covalently closed loops form right- or left-handed supercoils, depending on the sign of overtwist or undertwist. Short stem-loop structures melt at elevated temperatures and reanneal when the temperature is carefully lowered. Overall, most qualitative properties of real DNA arise naturally in the model from local interactions at the base-pair level.

Accommodating variety in iron-responsive elements: Crystal structure of transferrin receptor 1 B IRE bound to iron regulatory protein 1

Iron responsive elements (IREs) are short stem-loop structures found in several mRNAs encoding proteins involved in cellular iron metabolism. Iron regulatory proteins (IRPs) control iron homeostasis through differential binding to the IREs, accommodating any sequence or structural variations that the IREs may present. Here we report the structure of IRP1 in complex with transferrin receptor 1 B (TfR B) IRE, and compare it to the complex with ferritin H (Ftn H) IRE. The two IREs are bound to IRP1 through nearly identical protein-RNA contacts, although their stem conformations are significantly different. These results support the view that binding of different IREs with IRP1 depends both on protein and RNA conformational plasticity, adapting to RNA variation while retaining conserved protein-RNA contacts.

English

English

Characterization of RNA aptamers that disrupt the RUNX1–CBFβ/DNA complex

The transcription factor RUNX1 (AML1) is an important regulator of haematopoiesis, and an important fusion partner in leukaemic translocations. High-affinity DNA binding by RUNX1 requires the interaction of the RUNX1 Runt-Homology-Domain (RHD) with the core-binding factor β protein (CBFβ). To generate novel reagents for in vitro and in vivo studies of RUNX1 function, we have selected high-affinity RNA aptamers against a recombinant RHD–CBFβ complex. Selection yielded two sequence families, each dominated by a single consensus sequence. Aptamers from each family disrupt DNA binding by the RUNX1 protein in vitro and compete with sequence-specific dsDNA binding. Minimal, high-affinity (∼100–160 nM) active aptamer fragments 28 and 30 nts in length, consisting of simple short stem-loop structures, were then identified. These bind to the RHD subunit and disrupt its interaction with CBFβ, which is consistent with reduced DNA affinity in the presence of aptamer. These aptamers represent new reagents that target a novel surface on the RHD required to stabilize the recombinant RHD–CBFβ complex and thus will further aid exploring the functions of this key transcription factor.

Prediction of the structure of the common perimitochondrial localization signal of nuclear transcripts in yeast.

Many nuclear genes encoding mitochondrial proteins require specific localization of their mRNAs to the vicinity of mitochondria for proper expression. Studies in Saccharomyces cerevisiae have shown that the cis-acting signal responsible for subcellular localization of mRNAs is localized in the 3' UTR of the transcript. In this paper we present an in silico approach for prediction of a common perimitochondrial localization signal of nuclear transcripts encoding mitochondrial proteins. We computed a consensus structure for this signal by comparison of 3' UTR models for about 3000 yeast transcripts with known localization. Our studies show a short stem-loop structure which appears in most mRNAs localized to the vicinity of mitochondria. The degree of similarity of a given 3' UTR to our consensus structure strongly correlates with experimentally determined perimitochondrial localization of the mRNA, therefore we believe that the structure we predicted acts as a subcellular localization signal. Since our algorithm operates on structures, it seems to be more reliable than sequence-based algorithms. The good predictive value of our model is supported by statistical analysis.

Structure of the phylogenetically most conserved domain of SRP RNA

The signal recognition particle (SRP) is a phylogenetically conserved ribonucleoprotein required for cotranslational targeting of proteins to the membrane of the endoplasmic reticulum of the bacterial plasma membrane. Domain IV of SRP RNA consists of a short stem-loop structure with two internal loops that contain the most conserved nucleotides of the molecule. All known essential interactions of SRP occur in that moiety containing domain IV. The solution structure of a 43-nt RNA comprising the complete Escherichia coli domain IV was determined by multidimensional NMR and restrained molecular dynamics refinement. Our data confirm the previously determined rigid structure of a smaller subfragment containing the most conserved, symmetric internal loop A (Schmitz et al., Nat Struct Biol, 1999, 6:634-638), where all conserved nucleotides are involved in nucleotide-specific structural interactions. Asymmetric internal loop B provides a hinge in the RNA molecule; it is partially flexible, yet also uniquely structured. The longer strand of internal loop B extends the major groove by creating a ledge-like arrangement; for loop B however, there is no obvious structural role for the conserved nucleotides. The structure of domain IV suggests that loop A is the initial site for the RNA/protein interaction creating specificity, whereas loop B provides a secondary interaction site.

RNA recognition by the joint action of two nucleolin RNA-binding domains: genetic analysis and structural modeling.

The interaction of nucleolin with a short stem-loop structure (NRE) requires two contiguous RNA-binding domains (RBD 1+2). The structural basis for RNA recognition by these RBDs was studied using a genetic system in Escherichia coli. Within each of the two domains, we identified several mutations that severely impair interaction with the RNA target. Mutations that alter RNA-binding specificity were also isolated, suggesting the identity of specific contacts between RBD 1+2 amino acids and nucleotides within the NRE stem-loop. Our data indicate that both RBDs participate in a joint interaction with the NRE and that each domain uses a different surface to contact the RNA. The constraints provided by these genetic data and previous mutational studies have enabled us to propose a three-dimensional model of nucleolin RBD 1+2 bound to the NRE stem-loop.

Localization of nucleolin binding sites on human and mouse pre-ribosomal RNA.

Nucleolin, a major RNA binding protein of the nucleolus is found associated mainly to the pre-ribosomal particles and is absent from the cytoplasmic mature ribosomes. The role of this protein in ribosome biogenesis remains largely unknown, and is likely to be reflected by its RNA binding properties. Nucleolin contains in its central domain four RNA recognition motifs (RRM, also called RBD for RNA binding domain) which are conserved among different species. RNA binding studies have revealed that nucleolin interacts specifically with a short stem loop structure called NRE (nucleolin recognition element). We show that nucleolin extracted from human, hamster and mouse cells interacts with the same specificity and affinity to a mouse 5'ETS (external transcribed spacer) RNA fragment which contains a NRE motif. A similar structure within the human 5'ETS is also efficiently recognized by mouse nucleolin. We identified putative NRE not only in the 5'ETS but also in the 3'ETS, ITS (internal transcribed spacer) and in the 18S and 28S RNA sequences. This is in agreement with in vivo cross-linking data and a previous immunocytological analysis of ribosomal transcription units. Interestingly, we found that all the NRE localized in the 28S region are within the variable domains. Despite considerable sequence divergence of these domains, several of the NRE have sequences perfectly conserved between these two species. This suggests that these nucleolin binding sites might be functionally important, in particular for ribosome biogenesis.

Binding of the adenovirus VAI RNA to the interferon-induced 68-kDa protein kinase correlates with function.

In human cells infected with adenovirus, the virus-associated RNA VAI blocks the activation of the interferon-induced double-stranded-RNA-dependent 68-kDa protein kinase (p68) and maintains normal levels of protein synthesis at late times after infection. VAI antagonizes the kinase activity by binding to p68. The structure of VAI consists of two long, base-paired stems connected by a complex short stem-loop structure. Previous work using a series of adenovirus mutants showed that the structural determinants of the VAI RNA that are essential for function reside in the central complex short stem-loop structure and adjacent base-paired regions (functional domain); the long duplex regions were found to be dispensable for function. To determine whether binding of VAI to p68 correlates with function and whether the structural determinants that are essential for function are also essential for binding, we studied the interaction of wild-type and several mutant VAI RNAs with p68 in whole cells. The p68-VAI complexes from mutant- and wild-type-infected cells were immunoprecipitated by an anti-p68 monoclonal antibody. The mutant RNAs that functioned efficiently in the cells bound to p68 efficiently in the cells, whereas functionally impaired mutants failed to bind to p68, indicating that the binding of the VAI RNA to p68 correlates well with function. In vitro binding assays with immunopurified p68 confirmed these observations. Secondary-structure analysis of several mutant VAI RNAs suggests that the binding does not depend on the long duplex regions but requires all the elements of the functional domain. We propose that the functional domain and the p68-binding domain of the VAI RNA are identical.