RNA Secondary Structure Formation Research Articles

Denaturing and dNTPs reagents improve SARS-CoV-2 detection via single and multiplex RT-qPCR

Background The COVID-19 pandemic, caused by the SARS-CoV-2, can be effectively managed with diagnostic tools such as RT-qPCR. However, it can produce false-negative results due to viral mutations and RNA secondary structures from the target gene sequence. Methods With High Performance Computing, the complete SARS-CoV-2 genome was obtained from the GenBank/GISAID to generate consensus sequences to design primers/probes for RT-qPCR. ORF8 gene was selected, meanwhile, E and N and RNAse P were according to CDC protocol. Nasopharyngeal swab samples were collected from patients diagnosed with SARS-CoV-2. Total RNA was purified according MagMAX kit, it was used in single, and multiplex RT-qPCR. To avoid templated secondary structures, compensate nucleotide proportions and improve Ct values, a solution composed of tetraethylammonium chloride and dimethyl sulfoxide and other with corresponding to dNTPs proportions in accordance SARS-CoV-2 genome were included. Sensitivity and specificity according to Ct values were determined with the Caret package in R software. Results 126,576 SARS-CoV-2 genomes from January to December 2020 comprised a database. From this, a region near of 5′ ORF8 gene showed three stem-loops was used for primers/FAM-probe. 49 samples were obtained, from them, 22 were positive to gene selected regions. Interestingly, samples from October to November 2020 were positive for all regions, however, in January 2021 different results were observed in ORF8. An improvement in Ct with the adjuvant solutions was determined in all samples with others SARS-CoV-2 primers/probes, for both single and multiplex RT-qPCR. The inclusion of the denaturing solution in single RT-qPCR increased its sensitivity with respect to the commercial method, while in multiplex the opposite was generated. Conclusions Including adjuvant solutions to prevent the formation of RNA secondary structures and the adjustment of the nucleotide ratios of SARS-CoV-2 improved single and multiplex RT-qPCR for viral identification, demonstrating its potential application in health public.

Monitoring Real-Time Temperature Dynamics of a Short RNA Hairpin Using Förster Resonance Energy Transfer and Circular Dichroism.

RNA molecules play crucial roles in gene expression regulation and cellular signaling, and these functions are governed by the formation of RNA secondary and tertiary structures. These structures are highly dynamic and subject to rapid changes in response to environmental cues, temperature in particular. Thermosensitive RNA secondary structures have been harnessed by multiple organisms to survey their temperature environment and to adjust gene expression accordingly. It is thus highly desirable to observe RNA structural changes in real time over a range of temperatures. Multiple approaches have been developed to study structural dynamics, but many of these require extensive processing of the RNA, large amounts of RNA input, and/or cannot be applied under physiological conditions. Here, we describe the use of a dually fluorescently labeled RNA oligonucleotide (containing a predicted hairpin structure) to monitor subtle RNA structural dynamics in vitro by Förster resonance energy transfer (FRET) and circular dichroism (CD) spectroscopy. These approaches can be employed under physiologically relevant conditions over a range of temperatures and with RNA concentrations as low as 200nM; they enable us to observe RNA structural dynamics in real time and to correlate these dynamics with changes in biological processes such as translation.

Transcriptome-wide profiling of RNA N4-cytidine acetylation in Arabidopsis thaliana and Oryza sativa

Acetylation of N4-cytidine (ac4C) has recently been discovered as a novel modification of mRNA. RNA ac4C modification has been shown to be a key regulator of RNA stability, RNA translation, and the thermal stress response. However, its existence in eukaryotic mRNAs is still controversial. In plants, the existence, distribution pattern, and potential function of RNA ac4C modification are largely unknown. Here we report the presence of ac4C in the mRNAs of both Arabidopsis thaliana and rice (Oryza sativa). By comparing two ac4C sequencing methods, we found that RNA immunoprecipitation and sequencing (acRIP-seq), but not ac4C sequencing, was suitable for plant RNA ac4C sequencing. We present transcriptome-wide atlases of RNA ac4C modification in A. thaliana and rice mRNAs obtained by acRIP-seq. Analysis of the distribution of RNA ac4C modifications showed that ac4C is enriched near translation start sites in rice mRNAs and near translation start sites and translation end sites in Arabidopsis mRNAs. The RNA ac4C modification level is positively correlated with RNA half-life and the number of splicing variants. Similar to that in mammals, the translation efficiency of ac4C target genes is significantly higher than that of other genes. Our in vitro translation results confirmed that RNA ac4C modification enhances translation efficiency. We also found that RNA ac4C modification is negatively correlated with RNA structure. These results suggest that ac4C is a conserved mRNA modification in plants that contributes to RNA stability, splicing, translation, and secondary structure formation.

RAFFT: Efficient prediction of RNA folding pathways using the fast Fourier transform.

We propose a novel heuristic to predict RNA secondary structure formation pathways that has two components: (i) a folding algorithm and (ii) a kinetic ansatz. This heuristic is inspired by the kinetic partitioning mechanism, by which molecules follow alternative folding pathways to their native structure, some much faster than others. Similarly, our algorithm RAFFT starts by generating an ensemble of concurrent folding pathways ending in multiple metastable structures, which is in contrast with traditional thermodynamic approaches that find single structures with minimal free energies. When we constrained the algorithm to predict only 50 structures per sequence, near-native structures were found for RNA molecules of length ≤ 200 nucleotides. Our heuristic has been tested on the coronavirus frameshifting stimulation element (CFSE): an ensemble of 68 distinct structures allowed us to produce complete folding kinetic trajectories, whereas known methods require evaluating millions of sub-optimal structures to achieve this result. Thanks to the fast Fourier transform on which RAFFT (RNA folding Algorithm wih Fast Fourier Transform) is based, these computations are efficient, with complexity [Formula: see text].

Amino acid repeats avert mRNA folding through conservative substitutions and synonymous codons, regardless of codon bias

A significant number of proteins in all living species contains amino acid repeats (AARs) of various lengths and compositions, many of which play important roles in protein structure and function. Here, I have surveyed select homopolymeric single [(A)n] and double [(AB)n] AARs in the human proteome. A close examination of their codon pattern and analysis of RNA structure propensity led to the following set of empirical rules: (1) One class of amino acid repeats (Class I) uses a mixture of synonymous codons, some of which approximate the codon bias ratio in the overall human proteome; (2) The second class (Class II) disregards the codon bias ratio, and appears to have originated by simple repetition of the same codon (or just a few codons); and finally, (3) In all AARs (including Class I, Class II, and the in-betweens), the codons are chosen in a manner that precludes the formation of RNA secondary structure. It appears that the AAR genes have evolved by orchestrating a balance between codon usage and mRNA secondary structure. The insights gained here should provide a better understanding of AAR evolution and may assist in designing synthetic genes.

Single molecule identification of homology-dependent interactions between long ssRNA and dsDNA.

Long non-coding RNAs (lncRNAs) are prominently associated with chromosomes in an ever-increasing diversity of roles. To provide further insight into the potential nature of these associations, we have explored, for the first time, the interaction of long single-stranded (ss) RNAs with cognate homologous double-stranded (ds) DNA in vitro. Using magnetic tweezers, we measured the effects of ssRNA on force extension curves for dsDNA. We observe that the presence of ssRNA impedes the extension of dsDNA, specifically at low forces, dependent on homology between the RNA and DNA species, and dependent on ssRNA lengths (≥1 kb). The observed effect also depends on the concentration of ssRNA and is abolished by overstretching of the dsDNA. These findings show that significant homologous contacts can occur between long ssRNA and dsDNA in the absence of protein and that these contacts alter the mechanical properties of the dsDNA. We propose that long ssRNA interacts paranemically with long dsDNA via periodic short homologous interactions, e.g. mediated by RNA/DNA triplex-formation, and that dsDNA extension is impeded by formation of RNA secondary structure in the intervening unbound regions. Analogous interactions in vivo would permit lncRNAs to mediate the juxtaposition of two or more DNA regions on the same or different chromosomes.

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.

Entropic Determinants and Barriers in the Formation of RNA Secondary Structures

Any nucleic acid structure that contains single-stranded (ss) segments is dominated by entropy, but molecular-level information about these entropic determinants are inaccessible to direct experiments. A new Monte Carlo (MC) simulation has been developed based on a recently reported backbone conformational formula (BCV/-c) for RNAs. BCV/-c analytically enumerates all conformational states on the sugar-phosphate backbone. It has been validated rigorously against experiments, and the new MC algorithm, NUCLEIC, is atomistically accurate, computationally efficient, as well as predictive. We used NUCLEIC to investigate the entropic landscape of a comprehensive set of RNA secondary structures including: hairpins, stem-loops, symmetric and asymmetric bulges, 2-way, 3-way and 4-way junctions, as well as triplexes and quadruplexes. We found strong evidence that the kinetic barriers to the formation of many of these secondary structural elements are determined by the entropic bottlenecks associated with the unpaired ss tracts on each structure, though the thermodynamic free energy factor that stabilizes them come predominantly from base stacking. Based on these entropy data, we computed the rates of formation of various secondary structures and found that the kinetics of different pathways leading to the same product structure often have significantly different rates. These results highlight the importance of a molecular-level understanding of entropy as a defining factor behind RNA structures.

Reentrant melting of RNA with quenched sequence randomness.

The effect of quenched sequence disorder on the thermodynamics of RNA secondary structure formation is investigated for two- and four-letter alphabet models using the constrained annealing approach, from which the temperature behavior of the free energy, specific heat, and helicity is analytically obtained. For competing base pairing energies, the calculations reveal reentrant melting at low temperatures, in excellent agreement with numerical results. Our results suggest an additional mechanism for the experimental phenomenon of RNA cold denaturation.

Phase transition in random planar diagrams and RNA-type matching

We study the planar matching problem, defined by a symmetric random matrix with independent identically distributed entries, taking values zero and one. We show that the existence of a perfect planar matching structure is possible only above a certain critical density, p(c), of allowed contacts (i.e., of ones). Using a formulation of the problem in terms of Dyck paths and a matrix model of planar contact structures, we provide an analytical estimation for the value of the transition point, p(c), in the thermodynamic limit. This estimation is close to the critical value, p(c)≈0.379, obtained in numerical simulations based on an exact dynamical programming algorithm. We characterize the corresponding critical behavior of the model and discuss the relation of the perfect-imperfect matching transition to the known molten-glass transition in the context of random RNA secondary structure formation. In particular, we provide strong evidence supporting the conjecture that the molten-glass transition at T=0 occurs at p(c).

Unified approach to partition functions of RNA secondary structures

RNA secondary structure formation is a field of considerable biological interest as well as a model system for understanding generic properties of heteropolymer folding. This system is particularly attractive because the partition function and thus all thermodynamic properties of RNA secondary structure ensembles can be calculated numerically in polynomial time for arbitrary sequences and homopolymer models admit analytical solutions. Such solutions for many different aspects of the combinatorics of RNA secondary structure formation share the property that the final solution depends on differences of statistical weights rather than on the weights alone. Here, we present a unified approach to a large class of problems in the field of RNA secondary structure formation. We prove a generic theorem for the calculation of RNA folding partition functions. Then, we show that this approach can be applied to the study of the molten-native transition, denaturation of RNA molecules, as well as to studies of the glass phase of random RNA sequences.

Conformational stabilization of the membrane embedded targeting domain of the lysosomal peptide transporter TAPL for solution NMR

The ATP binding cassette transporter TAPL translocates cytosolic peptides into the lumen of lysosomes driven by the hydrolysis of ATP. Functionally, this transporter can be divided into coreTAPL, comprising the transport function, and an additional N-terminal transmembrane domain called TMD0, which is essential for lysosomal targeting and mediates the interaction with the lysosomal associated membrane proteins LAMP-1 and LAMP-2. To elucidate the structure of this unique domain, we developed protocols for the production of high quantities of cell-free expressed TMD0 by screening different N-terminal expression tags. Independently of the amino acid sequence, high expression was detected for AU-rich sequences in the first seven codons, decreasing the free energy of RNA secondary structure formation at translation initiation. Furthermore, avoiding NGG codons in the region of translation initiation demonstrated a positive effect on expression. For NMR studies, conditions were optimized for high solubilization efficiency, long-term stability, and high quality spectra. A most critical step was the careful exchange of the detergent used for solubilization by the detergent dihexanoylphosphatidylcholine. Several constructs of different size were tested in order to stabilize the fold of TMD0 as well as to reduce the conformation exchange. NMR spectra with sufficient resolution and homogeneity were finally obtained with a TMD0 derivative only modified by a C-terminal His10-tag and containing a codon optimized AT-rich sequence.

Mechanistic insights into mutually exclusive splicing in dynamin 1

Mutually exclusive splicing is a strictly regulated pattern of alternative splicing. A specific group of mutually exclusive splicing events has been shown to be regulated by the formation of specific RNA secondary structures. This type of regulation has been shown to exist only in arthropods. The present study involved a detailed sequence analysis of human gene structures that undergo mutually exclusive splicing, which showed that this type of regulation may also occur in dynamin 1 in mammals. A phylogenetic analysis revealed that the dynamin 1 orthologs in invertebrates did not share the same sequence features, which suggests that the regulatory mechanism has independently evolved in the mammalian lineage. Therefore, the emergence of this elaborate mechanism for mutually exclusive splicing may be attributable to mechanistic convergence.

Combinatorics of locally optimal RNA secondary structures

It is a classical result of Stein and Waterman that the asymptotic number of RNA secondary structures is 1.104366∙n-3/2∙2.618034n. Motivated by the kinetics of RNA secondary structure formation, we are interested in determining the asymptotic number of secondary structures that are locally optimal, with respect to a particular energy model. In the Nussinov energy model, where each base pair contributes -1 towards the energy of the structure, locally optimal structures are exactly the saturated structures, for which we have previously shown that asymptotically, there are 1.07427∙n-3/2∙2.35467n many saturated structures for a sequence of length n. In this paper, we consider the base stacking energy model, a mild variant of the Nussinov model, where each stacked base pair contributes -1 toward the energy of the structure. Locally optimal structures with respect to the base stacking energy model are exactly those secondary structures, whose stems cannot be extended. Such structures were first considered by Evers and Giegerich, who described a dynamic programming algorithm to enumerate all locally optimal structures. In this paper, we apply methods from enumerative combinatorics to compute the asymptotic number of such structures. Additionally, we consider analogous combinatorial problems for secondary structures with annotated single-stranded, stacking nucleotides (dangles).

Comparison of the Roles of Nucleotide Synthesis, Polymerization, and Recombination in the Origin of Autocatalytic Sets of RNAs

Ribozymes that act as polymerases and nucleotide synthases are known experimentally, even though no fully self-replicating system has yet been found. If the RNA World hypothesis is true, ribozymes must have arisen initially from within a random abiotic polymerization system. To investigate the origin of the RNA world, we studied a mathematical model of a chemical reaction system describing RNA polymerization. It is supposed that, in absence of ribozymes, polymerization occurs at a small spontaneous rate, and that in the presence of polymerase ribozymes, polymerization occurs at a faster rate that is proportional to the ribozyme concentration. Chains must be longer than a minimum threshold length in order to have the possibility of acting as ribozymes. The reaction system has two stable states that we term dead and living. The dead state is controlled by the small spontaneous rate and has negligible concentration of ribozymes. The living state has high concentration of ribozymes, and the reaction rates are determined by the ribozymes; thus, the system is autocatalytic. Concentration fluctuations in a finite volume can cause a transition to occur from the dead to the living state, that is, an origin of life occurs within this model. We also consider ribozymes that catalyze nucleotide synthesis. We show that living and dead states arise in the presence of synthase ribozymes in the same way as for polymerases. It has been proposed that recombination reactions are a way of generating long RNA chains in the early stages of life. We show that if the possibility of random reversible recombination reactions is added to our model, this does not lead to an increase in long polymer concentration. Thus, if recombination is fully reversible, there is no autocatalytic state controlled by recombination. Nevertheless, recombination can play an important role in ribozyme synthesis if there is an additional process that keeps the recombination reactions out of equilibrium. We modeled a case studied experimentally in which building block strands of moderate length associate due to RNA secondary structure formation. A recombination reaction then occurs between these strands to form a longer sequence that catalyzes its own formation via the recombination reaction. This system has an autocatalytic state, and it is possible for it to arise within our random polymerization system. If complexes formed by associations of shorter strands can act as catalysts without the requirement that the strands be covalently linked, this would alleviate the need for synthesis of very long strands; hence, it makes the emergence of an autocatalytic system from an abiotic random polymerization system much more likely.

Association of TLR7 variants with AIDS-like disease and AIDS vaccine efficacy in rhesus macaques.

In HIV infection, TLR7-triggered IFN-α production exerts a direct antiviral effect through the inhibition of viral replication, but may also be involved in immune pathogenesis leading to AIDS. TLR7 could also be an important mediator of vaccine efficacy. In this study, we analyzed polymorphisms in the X-linked TLR7 gene in the rhesus macaque model of AIDS. Upon resequencing of the TLR7 gene in 36 rhesus macaques of Indian origin, 12 polymorphic sites were detected. Next, we identified three tightly linked single nucleotide polymorphisms (SNP) as being associated with survival time. Genotyping of 119 untreated, simian immunodeficiency virus (SIV)-infected male rhesus macaques, including an ‘MHC adjusted’ subset, revealed that the three TLR7 SNPs are also significantly associated with set-point viral load. Surprisingly, this effect was not observed in 72 immunized SIV-infected male monkeys. We hypothesize (i) that SNP c.13G>A in the leader peptide is causative for the observed genotype-phenotype association and that (ii) the underlying mechanism is related to RNA secondary structure formation. Therefore, we investigated a fourth SNP (c.-17C>T), located 17 bp upstream of the ATG translation initiation codon, that is also potentially capable of influencing RNA structure. In c.13A carriers, neither set-point viral load nor survival time were related to the c.-17C>T genotype. In c.13G carriers, by contrast, the c.-17C allele was significantly associated with prolonged survival. Again, no such association was detected among immunized SIV-infected macaques. Our results highlight the dual role of TLR7 in immunodeficiency virus infection and vaccination and imply that it may be important to control human AIDS vaccine trials, not only for MHC genotype, but also for TLR7 genotype.

Ultraconserved cDNA segments in the human transcriptome exhibit resistance to folding and implicate function in translation and alternative splicing

Ultraconservation, defined as perfect human-to-rodent sequence identity at least 200-bp long, is a strong indicator of evolutionary and functional importance and has been explored extensively at the genome level. However, it has not been investigated at the transcript level, where such extreme conservation might highlight loci with important post-transcriptional regulatory roles. We present 96 ultraconserved cDNA segments (UCSs), stretches of human mature mRNAs that match identically with orthologous regions in the mouse and rat genomes. UCSs can span multiple exons, a feature we leverage here to elucidate the role of ultraconservation in post-transcriptional regulation. UCS sites are implicated in functions at essentially every post-transcriptional stage: pre-mRNA splicing and degradation through alternative splicing and nonsense-mediated decay (AS-NMD), mature mRNA silencing by miRNA, fast mRNA decay rate and translational repression by upstream AUGs. We also found UCSs to exhibit resistance to formation of RNA secondary structure. These multiple layers of regulation underscore the importance of the UCS-containing genes as key global RNA processing regulators, including members of the serine/arginine-rich protein and heterogeneous nuclear ribonucleoprotein (hnRNP) families of essential splicing regulators. The discovery of UCSs shed new light on the multifaceted, fine-tuned and tight post-transcriptional regulation of gene families as conserved through the majority of the mammalian lineage.

The DEAD-box protein PMH2 is required for efficient group II intron splicing in mitochondria of Arabidopsis thaliana

In Arabidopsis thaliana the putative mitochondrial RNA helicases PMH1 and PMH2 are members of the large DEAD-box protein family. Our previous characterization of these proteins revealed that PMH1 and/or PMH2 are part of high molecular weight complexes. Now T-DNA insertion lines were established and characterized for each of these genes. Immunodetection analysis of cell suspension cultures established from pmh1-1 and pmh2-1 mutants revealed that indeed both DEAD-box proteins are detectable in large protein complexes with PMH2 being much more abundant than PMH1. In plants the knockout of PMH2 leads to reduced group II intron splicing efficiency. In addition the steady-state levels of several mature mitochondrial mRNAs are decreased while transcription is not influenced. This molecular phenotype suggests that PMH2 acts at the posttranscriptional level with a potential function as RNA chaperone required for formation or maintenance of complex RNA secondary structures of introns rather than a direct role in splicing. In contrast, the investigation of a pmh1-1 knockout line did not reveal any influence of this protein on processing and abundance of mitochondrial transcripts.

Locomotif: from graphical motif description to RNA motif search

Motivated by the recent rise of interest in small regulatory RNAs, we present Locomotif--a new approach for locating RNA motifs that goes beyond the previous ones in three ways: (1) motif search is based on efficient dynamic programming algorithms, incorporating the established thermodynamic model of RNA secondary structure formation. (2) motifs are described graphically, using a Java-based editor, and search algorithms are derived from the graphics in a fully automatic way. The editor allows us to draw secondary structures, annotated with size and sequence information. They closely resemble the established, but informal way in which RNA motifs are communicated in the literature. Thus, the learning effort for Locomotif users is minimal. (3) Locomotif employs a client-server approach. Motifs are designed by the user locally. Search programs are generated and compiled on a bioinformatics server. They are made available both for execution on the server, and for download as C source code plus an appropriate makefile. Locomotif is available at http://bibiserv.techfak.uni-bielefeld.de/locomotif.

SREBP regulates translation by promoter selection and formation of RNA secondary structure under anoxia in fission yeast

SREBP regulates translation by promoter selection and formation of RNA secondary structure under anoxia in fission yeast