RNA Base Pairs Research Articles

High Throughput Characterization of RNA Tertiary Elements

The function of many RNAs is intimately linked to the final three-dimensional conformation of the folded molecule. Folding of an RNA can be viewed as hierarchical: RNA secondary structure is the formation of locally ordered helices from a given primary sequence of bases, and RNA tertiary structure is the reduction of conformational space explored by local helices through the formation of long-range contacts. The thermodynamic principles guiding the formation of RNA secondary structure are largely successful at predicting local base pairing of RNA. However, RNA tertiary structure remains difficult to predict, largely because of a dearth of data on the conformational preferences of RNA junctions and helices. By making thermodynamic measurements on millions of different RNAs in parallel, our work begins to dissect the energetics of RNA tertiary structure formation. In a discrete model system, we determined simplifying rules for conformational preferences of helices and junctions.

A DEAD-box RNA helicase promotes thermodynamic equilibration of kinetically trapped RNA structures in vivo.

RNAs must assemble into specific structures in order to carry out their biological functions, but in vitro RNA folding reactions produce multiple misfolded structures that fail to exchange with functional structures on biological time scales. We used carefully designed self-cleaving mRNAs that assemble through well-defined folding pathways to identify factors that differentiate intracellular and in vitro folding reactions. Our previous work showed that simple base-paired RNA helices form and dissociate with the same rate and equilibrium constants in vivo and in vitro. However, exchange between adjacent secondary structures occurs much faster in vivo, enabling RNAs to quickly adopt structures with the lowest free energy. We have now used this approach to probe the effects of an extensively characterized DEAD-box RNA helicase, Mss116p, on a series of well-defined RNA folding steps in yeast. Mss116p overexpression had no detectable effect on helix formation or dissociation kinetics or on the stability of interdomain tertiary interactions, consistent with previous evidence that intracellular factors do not affect these folding parameters. However, Mss116p overexpression did accelerate exchange between adjacent helices. The nonprocessive nature of RNA duplex unwinding by DEAD-box RNA helicases is consistent with a branch migration mechanism in which Mss116p lowers barriers to exchange between otherwise stable helices by the melting and annealing of one or two base pairs at interhelical junctions. These results suggest that the helicase activity of DEAD-box proteins like Mss116p distinguish intracellular RNA folding pathways from nonproductive RNA folding reactions in vitro and allow RNA structures to overcome kinetic barriers to thermodynamic equilibration in vivo.

Correlations of NBO energies of individual hydrogen bonds in nucleic acid base pairs with some QTAIM parameters

DNA and RNA base-pairs are the most important systems containing multiple hydrogen bonds. Characterizing the energy of individual intermolecular interactions in such systems is vital and still an open problem that has been tackled here within the framework of the natural bond orbital (NBO) and the quantum theory of atoms in molecules (QTAIM) theories. In the NBO language, energy of an individual H-bond depends on the interaction of the n Y, σ*XH, and σXH orbitals directly involved in H-bonding. A partial charge transfer between donor (n Y) and acceptor (σ*XH) orbitals provides a substantial bonding contribution to the energies of the H-bonds, and in the end, the H-bonded complexes. It is accompanied with a repulsive contribution due to the proximity of the n Y and σXH orbitals. Energies of the individual H-bonds, resulting from addition of the both terms, were correlated with several parameters, provided by the QTAIM analysis which has also been extensively used to characterize the hydrogen bond. The calculations were performed for the G–C and A–T Watson–Crick base pairs, their substituted derivatives (by one of two substituents, NH3 + or OH2 +), A–U occurring in RNA and a wobble pair G–U. The best correlations were found for the NBO energy with the electron density and the potential energy density at H-bond critical points. The correlations held for the heterogeneous samples of HBs of different types, i.e. N–H···O, N–H···N, and C–H···O, occurring simultaneously in DNA base pairs.

ChemInform Abstract: The “Speedy” Synthesis of Atom‐Specific 15N Imino/Amido‐Labeled RNA

AbstractReview: 82 refs.

New in silico approach to assessing RNA secondary structures with non-canonical base pairs.

BackgroundThe function of RNA is strongly dependent on its structure, so an appropriate recognition of this structure, on every level of organization, is of great importance. One particular concern is the assessment of base-base interactions, described as the secondary structure, the knowledge of which greatly facilitates an interpretation of RNA function and allows for structure analysis on the tertiary level. The RNA secondary structure can be predicted from a sequence using in silico methods often adjusted with experimental data, or assessed from 3D structure atom coordinates. Computational approaches typically consider only canonical, Watson-Crick and wobble base pairs. Handling of non-canonical interactions, important for a full description of RNA structure, is still very difficult.ResultsWe introduce our novel approach to assessing an extended RNA secondary structure, which characterizes both canonical and non-canonical base pairs, along with their type classification. It is based on predicting the RNA 3D structure from a user-provided sequence or a secondary structure that only describes canonical base pairs, and then deriving the extended secondary structure from atom coordinates. In our example implementation, this was achieved by integrating the functionality of two fully automated, high fidelity methods in a computational pipeline: RNAComposer for the 3D RNA structure prediction and RNApdbee for base-pair annotation.ConclusionsThe presented methodology ties together existing applications for RNA 3D structure prediction and base-pair annotation. The example performance, applying RNAComposer and RNApdbee, reveals better accuracy in non-canonical base pair assessment than the compared methods that directly predict RNA secondary structure.Electronic supplementary materialThe online version of this article (doi:10.1186/s12859-015-0718-6) contains supplementary material, which is available to authorized users.

A New Role for an Old Drug: Metformin Targets MicroRNAs in Treating Diabetes and Cancer.

MicroRNAs (miRNAs) are a family of short, noncoding, 19-23 base pair RNA molecules. Due to their unique role in gene regulation in various tissues, miRNAs play important roles in regulating insulin secretion, metabolic disease, and cancer biology. Emerging evidence demonstrates that miRNAs could also be novel diagnostic markers for a variety of disease states. Additionally, miRNAs have been found to function either as oncogenes, or tumor suppressor genes in cerian cancers. An increasing number of studies have been conducted investigating new drugs targeting miRNAs as a potential anticancer therapy. Metformin is the most widely prescribed medication for treating Type 2 diabetes (T2D). Recent clinical data suggests that metformin impacts the miRNA profile in T2D subjects. Most excitingly, studies have found that metformin is protective against cancer. The anticancer activity of metformin is mediated through a direct regulation of miRNAs, which further modulates several downstream genes in metabolic or preoncogenic pathways. These miRNAs are, therefore, prospective therapeutic targets for treating diabetes and cancer which is the topic of this review. Further study on the regulation of miRNAs by metformin could result in novel therapeutic strategies for recurrent or drug-esistant cancer, and as part of combinatorial approaches with conventional anticancer therapies.

Abstract 3993: MicroRNA analysis of aromatase inhibitor-resistant breast cancer cells reveals upregulation of a unique miRNA cluster on chromosome 14

Abstract Estrogen signaling has been shown to play a large role in the development and progression of breast tumors that express the estrogen receptor (ERα). Thus, endocrine therapies that either inhibit ERα action (tamoxifen or fulvestrant) or inhibit estrogen synthesis (aromatase inhibitors) have been increasingly valuable for the management of ER+ breast cancer. A frequent obstacle, however, is acquired resistance to endocrine therapies, often characterized by aberrant ERα expression and heightened growth factor signaling. Another aspect of acquired endocrine resistance that is now being examined is the role of microRNA (miRNA). miRNAs are 18-22 base pair RNA molecules whose primary role is to inhibit expression of mRNA targets by inhibition of translation or by degradation of the mRNA. Here, we have conducted next generation sequencing of the miRNA from three breast cancer cell lines - one ER+ line that is estrogen-dependent and AI-sensitive (MCF-7), and two ER+ long-term estrogen deprived (LTED) models of AI resistance (MCF-7:5C and MCF-7:2A). We found that in LTED/AI-resistant MCF-7:5C and MCF-7:2A cells there were over 330 and 240 miRNAs differentially expressed, respectively, compared to AI-sensitive MCF-7 breast cancer cells. Initially, we used qPCR to validate the expression of miRNAs that that were differentially expressed in both LTED cell lines vs. MCF-7. Notably, LTED cell lines had upregulated miR-181a (> 4 to 8-fold) and downregulated miR-221/222 expression compared to MCF-7. Elevated miR-181a has been shown to be associated with more aggressive breast cancers whereas miR-221/222 play a role in tamoxifen resistance through targeting ERα. Further bioinformatic analysis also revealed that the LTED cells had very unique expression profiles compared to one another, indicating that they have adapted to an estrogen-depleted environment through potentially different mechanisms. In MCF-7:5C cells, which have lost expression of the progesterone receptor (PR), a miRNA cluster on chromosome 14 is upregulated. This upregulated cluster, which encodes miR-127, miR-654, miR-889 and others, has been shown to be associated with Luminal B (ER+/PR-) breast cancer, consistent with the hormone receptor status of the MCF-7:5C cells. The oncogenic, estrogen-responsive miR-17/92 cluster on chromosome 13 is upregulated in the MCF-7:2A cells and these miRNAs target proteins including PTEN, p21, and Bim. Overall, these findings reveal altered expression of miRNAs in AI-resistant breast cancer cells and they suggest that targeting these miRNAs might be an effective strategy to reverse AI resistance in breast cancer patients. Citation Format: Erin L. Hayes, Jennifer Knapp, Joan Lewis-Wambi. MicroRNA analysis of aromatase inhibitor-resistant breast cancer cells reveals upregulation of a unique miRNA cluster on chromosome 14. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3993. doi:10.1158/1538-7445.AM2015-3993

An atlas of RNA base pairs involving modified nucleobases with optimal geometries and accurate energies.

Posttranscriptional modifications greatly enhance the chemical information of RNA molecules, contributing to explain the diversity of their structures and functions. A significant fraction of RNA experimental structures available to date present modified nucleobases, with half of them being involved in H-bonding interactions with other bases, i.e. ‘modified base pairs’. Herein we present a systematic investigation of modified base pairs, in the context of experimental RNA structures. To this end, we first compiled an atlas of experimentally observed modified base pairs, for which we recorded occurrences and structural context. Then, for each base pair, we selected a representative for subsequent quantum mechanics calculations, to find out its optimal geometry and interaction energy. Our structural analyses show that most of the modified base pairs are non Watson–Crick like and are involved in RNA tertiary structure motifs. In addition, quantum mechanics calculations quantify and provide a rationale for the impact of the different modifications on the geometry and stability of the base pairs they participate in.

The "Speedy" Synthesis of Atom-Specific (15)N Imino/Amido-Labeled RNA.

Although numerous reports on the synthesis of atom-specific (15)N-labeled nucleosides exist, fast and facile access to the corresponding phosphoramidites for RNA solid-phase synthesis is still lacking. This situation represents a severe bottleneck for NMR spectroscopic investigations on functional RNAs. Here, we present optimized procedures to speed up the synthesis of (15)N(1) adenosine and (15)N(1) guanosine amidites, which are the much needed counterparts of the more straightforward-to-achieve (15)N(3) uridine and (15)N(3) cytidine amidites in order to tap full potential of (1)H/(15)N/(15)N-COSY experiments for directly monitoring individual Watson-Crick base pairs in RNA. Demonstrated for two preQ1 riboswitch systems, we exemplify a versatile concept for individual base-pair labeling in the analysis of conformationally flexible RNAs when competing structures and conformational dynamics are encountered.

Light‐Triggered RNA Annealing by an RNA Chaperone

AbstractNon‐coding antisense RNAs regulate bacterial genes in response to nutrition or environmental stress, and can be engineered for artificial gene control. The RNA chaperone Hfq accelerates antisense pairing between non‐coding RNAs and their mRNA targets, by a mechanism still unknown. We used a photocaged guanosine derivative in an RNA oligonucleotide to temporally control Hfq catalyzed annealing. Using a fluorescent molecular beacon as a reporter, we observed RNA duplex formation within 15 s following irradiation (3 s) of photocaged RNA complexed with Hfq. The results showed that the Hfq chaperone directly stabilizes the initiation of RNA base pairs, and suggests a strategy for light‐activated control of gene expression by non‐coding RNAs.

Light-Triggered RNA Annealing by an RNA Chaperone.

Non-coding antisense RNAs regulate bacterial genes in response to nutrition or environmental stress, and can be engineered for artificial gene control. The RNA chaperone Hfq accelerates antisense pairing between non-coding RNAs and their mRNA targets, by a mechanism still unknown. We used a photocaged guanosine derivative in an RNA oligonucleotide to temporally control Hfq catalyzed annealing. Using a fluorescent molecular beacon as a reporter, we observed RNA duplex formation within 15 s following irradiation (3 s) of photocaged RNA complexed with Hfq. The results showed that the Hfq chaperone directly stabilizes the initiation of RNA base pairs, and suggests a strategy for light-activated control of gene expression by non-coding RNAs.

Biogenesis pathways of RNA guides in archaeal and bacterial CRISPR-Cas adaptive immunity.

CRISPR-Cas is an RNA-mediated adaptive immune system that defends bacteria and archaea against mobile genetic elements. Short mature CRISPR RNAs (crRNAs) are key elements in the interference step of the immune pathway. A CRISPR array composed of a series of repeats interspaced by spacer sequences acquired from invading mobile genomes is transcribed as a precursor crRNA (pre-crRNA) molecule. This pre-crRNA undergoes one or two maturation steps to generate the mature crRNAs that guide CRISPR-associated (Cas) protein(s) to cognate invading genomes for their destruction. Different types of CRISPR-Cas systems have evolved distinct crRNA biogenesis pathways that implicate highly sophisticated processing mechanisms. In Types I and III CRISPR-Cas systems, a specific endoribonuclease of the Cas6 family, either standalone or in a complex with other Cas proteins, cleaves the pre-crRNA within the repeat regions. In Type II systems, the trans-acting small RNA (tracrRNA) base pairs with each repeat of the pre-crRNA to form a dual-RNA that is cleaved by the housekeeping RNase III in the presence of the protein Cas9. In this review, we present a detailed comparative analysis of pre-crRNA recognition and cleavage mechanisms involved in the biogenesis of guide crRNAs in the three CRISPR-Cas types.

How do base-pairing small RNAs evolve?

The increasing numbers of characterized base-pairing small RNAs (sRNAs) and the identification of these regulators in a broad range of bacteria are allowing comparisons between species and explorations of sRNA evolution. In this review, we describe some examples of trans-encoded base-pairing sRNAs that are species-specific and others that are more broadly distributed. We also describe examples of sRNA orthologs where different features are conserved. These examples provide the background for a discussion of mechanisms of sRNA evolution and selective pressures on the sRNAs and their mRNA target(s).

Stacking geometry for non-canonical G:U wobble base pair containing dinucleotide sequences in RNA: dispersion-corrected DFT-D study.

Emergence of thousands of crystal structures of noncoding RNA molecules indicates its structural and functional diversity. RNA function is based upon a large variety of structural elements which are specifically assembled in the folded molecules. Along with the canonical Watson-Crick base pairs, different orientations of the bases to form hydrogen-bonded non-canonical base pairs have also been observed in the available RNA structures. Frequencies of occurrences of different non-canonical base pairs in RNA indicate their important role to maintain overall structure and functions of RNA. There are several reports on geometry and energetic stabilities of these non-canonical base pairs. However, their stacking geometry and stacking stability with the neighboring base pairs are not well studied. Among the different non-canonical base pairs, the G:U wobble base pair (G:U W:WC) is most frequently observed in the RNA double helices. Using quantum chemical method and available experimental data set we have studied the stacking geometry of G:U W:WC base pair containing dinucleotide sequences in roll-slide parameters hyperspace for different values of twist. This study indicates that the G:U W:WC base pair can stack well with the canonical base pairs giving rise to large interaction energy. The overall preferred stacking geometry in terms of roll, twist and slide for the eleven possible dinucleotide sequences is seen to be quite dependent on their sequences.

Stabilization of a Bimolecular Triplex by 3'-S-Phosphorothiolate Modifications: An NMR and UV Thermal Melting Investigation.

Triplexes formed from oligonucleic acids are key to a number of biological processes. They have attracted attention as molecular biology tools and as a result of their relevance in novel therapeutic strategies. The recognition properties of single-stranded nucleic acids are also relevant in third-strand binding. Thus, there has been considerable activity in generating such moieties, referred to as triplex forming oligonucleotides (TFOs). Triplexes, composed of Watson-Crick (W-C) base-paired DNA duplexes and a Hoogsteen base-paired RNA strand, are reported to be more thermodynamically stable than those in which the third strand is DNA. Consequently, synthetic efforts have been focused on developing TFOs with RNA-like structural properties. Here, the structural and stability studies of such a TFO, composed of deoxynucleic acids, but with 3'-S-phosphorothiolate (3'-SP) linkages at two sites is described. The modification results in an increase in triplex melting temperature as determined by UV absorption measurements. (1) H NMR analysis and structure generation for the (hairpin) duplex component and the native and modified triplexes revealed that the double helix is not significantly altered by the major groove binding of either TFO. However, the triplex involving the 3'-SP modifications is more compact. The 3'-SP modification was previously shown to stabilise G-quadruplex and i-motif structures and therefore is now proposed as a generic solution to stabilising multi-stranded DNA structures.

RNA World research-still evolving.

RNA World research-still evolving.

The DEAH-box helicase Dhr1 dissociates U3 from the pre-rRNA to promote formation of the central pseudoknot.

In eukaryotes, the highly conserved U3 small nucleolar RNA (snoRNA) base-pairs to multiple sites in the pre-ribosomal RNA (pre-rRNA) to promote early cleavage and folding events. Binding of the U3 box A region to the pre-rRNA is mutually exclusive with folding of the central pseudoknot (CPK), a universally conserved rRNA structure of the small ribosomal subunit essential for protein synthesis. Here, we report that the DEAH-box helicase Dhr1 (Ecm16) is responsible for displacing U3. An active site mutant of Dhr1 blocked release of U3 from the pre-ribosome, thereby trapping a pre-40S particle. This particle had not yet achieved its mature structure because it contained U3, pre-rRNA, and a number of early-acting ribosome synthesis factors but noticeably lacked ribosomal proteins (r-proteins) that surround the CPK. Dhr1 was cross-linked in vivo to the pre-rRNA and to U3 sequences flanking regions that base-pair to the pre-rRNA including those that form the CPK. Point mutations in the box A region of U3 suppressed a cold-sensitive mutation of Dhr1, strongly indicating that U3 is an in vivo substrate of Dhr1. To support the conclusions derived from in vivo analysis we showed that Dhr1 unwinds U3-18S duplexes in vitro by using a mechanism reminiscent of DEAD box proteins.

Stereospecificity of oligonucleotide interactions revisited: no evidence for heterochiral hybridization and ribozyme/DNAzyme activity.

A major challenge for the application of RNA- or DNA-oligonucleotides in biotechnology and molecular medicine is their susceptibility to abundant nucleases. One intriguing possibility to tackle this problem is the use of mirror-image (l-)oligonucleotides. For aptamers, this concept has successfully been applied to even develop therapeutic agents, so-called Spiegelmers. However, for technologies depending on RNA/RNA or RNA/DNA hybridization, like antisense or RNA interference, it has not been possible to use mirror-image oligonucleotides because Watson-Crick base pairing of complementary strands is (thought to be) stereospecific. Many scientists consider this a general principle if not a dogma. A recent publication proposing heterochiral Watson-Crick base pairing and sequence-specific hydrolysis of natural RNA by mirror-image ribozymes or DNAzymes (and vice versa) prompted us to systematically revisit the stereospecificity of oligonucleotides hybridization and catalytic activity. Using hyperchromicity measurements we demonstrate that hybridization only occurs among homochiral anti-parallel complementary oligonucleotide strands. As expected, achiral PNA hybridizes to RNA and DNA irrespective of their chirality. In functional assays we could not confirm an alleged heterochiral hydrolytic activity of ribozymes or DNAzymes. Our results confirm a strict stereospecificity of oligonucleotide hybridization and clearly argue against the possibility to use mirror-image oligonucleotides for gene silencing or antisense applications.

The role of N7 protonation of guanine in determining the structure, stability and function of RNA base pairs.

The roles of protonated nucleobases in stabilizing different structural motifs and in facilitating catalytic functions of RNA are well known. Among different polar sites of all the nucleobases, N7 of guanine has the highest protonation propensity at physiological pH. However, unlike other easily protonable sites such as N1 and N3 of adenine or N3 of cytosine, N7 protonation of guanine does not lead to the stabilization of base pairs involving its protonated Hoogsteen edge. It also does not facilitate its participation in any acid-base catalysis process. To explore the possible roles of N7 protonated guanine, we have studied its base pairing potentials involving WatsonCrick and sugar edges, which undergo major charge redistribution upon N7 protonation. We have carried out quantum chemical geometry optimization at the M05-2X/6-311G+(2d,2p) level, followed by interaction energy calculation at the MP2/aug-cc-pVDZ level, along with the analysis of the context of occurrence for selected base pairs involving the sugar edge or the WatsonCrick edge of guanine within a non-redundant set of 167 RNA crystal structures. Our results suggest that, four base pairs - G:C W:W trans, G:rC W:S cis, G:G W:H cis and G:G S:H trans may involve N7 protonated guanine. These base pairs deviate significantly from their respective experimental geometries upon QM optimization, but they retain their experimental geometries if guanine N7 protonation is considered during optimization. Our study also reveals the role of guanine N7 protonation (i) in stabilizing important RNA structural motifs, (ii) in providing a framework for designing pH driven molecular motors and (iii) in providing an alternative strategy to mimic the effect of post-transcriptional changes.

Profiling small RNA reveals multimodal substructural signals in a Boltzmann ensemble.

As the biomedical impact of small RNAs grows, so does the need to understand competing structural alternatives for regions of functional interest. Suboptimal structure analysis provides significantly more RNA base pairing information than a single minimum free energy prediction. Yet computational enhancements like Boltzmann sampling have not been fully adopted by experimentalists since identifying meaningful patterns in this data can be challenging. Profiling is a novel approach to mining RNA suboptimal structure data which makes the power of ensemble-based analysis accessible in a stable and reliable way. Balancing abstraction and specificity, profiling identifies significant combinations of base pairs which dominate low-energy RNA secondary structures. By design, critical similarities and differences are highlighted, yielding crucial information for molecular biologists. The code is freely available via http://gtfold.sourceforge.net/profiling.html.