Changes In RNA Secondary Structure Research Articles

Chikungunya Virus RNA Secondary Structures Impact Defective Viral Genome Production.

Chikungunya virus (CHIKV) is a mosquito-borne RNA virus that poses an emerging threat to humans. In a manner similar to other RNA viruses, CHIKV encodes an error-prone RNA polymerase which, in addition to producing full-length genomes, gives rise to truncated, non-functional genomes, which have been coined defective viral genomes (DVGs). DVGs have been intensively studied in the context of therapy, as they can inhibit viral replication and dissemination in their hosts. In this work, we interrogate the influence of viral RNA secondary structures on the production of CHIKV DVGs. We experimentally map RNA secondary structures of the CHIKV genome using selective 2'-hydroxyl acylation analyzed by primer extension and mutational profiling (SHAPE-MaP), which couples chemical labelling with next-generation sequencing. We correlate the inferred secondary structure with preferred deletion sites of CHIKV DVGs. We document an increased probability of DVG generation with truncations at unpaired nucleotides within the secondary structure. We then generated a CHIKV mutant bearing synonymous changes at the nucleotide level to disrupt the existing RNA secondary structure (CHIKV-D2S). We show that CHIKV-D2S presents altered DVG generation compared to wild-type virus, correlating with the change in RNA secondary structure obtained by SHAPE-MaP. Our work thus demonstrates that RNA secondary structure impacts CHIKV DVG production during replication.

The influence of 4-thiouridine labeling on pre-mRNA splicing outcomes.

Metabolic labeling is a widely used tool to investigate different aspects of pre-mRNA splicing and RNA turnover. The labeling technology takes advantage of native cellular machineries where a nucleotide analog is readily taken up and incorporated into nascent RNA. One such analog is 4-thiouridine (4sU). Previous studies demonstrated that the uptake of 4sU at elevated concentrations (>50μM) and extended exposure led to inhibition of rRNA synthesis and processing, presumably induced by changes in RNA secondary structure. Thus, it is possible that 4sU incorporation may also interfere with splicing efficiency. To test this hypothesis, we carried out splicing analyses of pre-mRNA substrates with varying levels of 4sU incorporation (0-100%). We demonstrate that increased incorporation of 4sU into pre-mRNAs decreased splicing efficiency. The overall impact of 4sU labeling on pre-mRNA splicing efficiency negatively correlates with the strength of splice site signals such as the 3' and the 5' splice sites. Introns with weaker splice sites are more affected by the presence of 4sU. We also show that transcription by T7 polymerase and pre-mRNA degradation kinetics were impacted at the highest levels of 4sU incorporation. Increased incorporation of 4sU caused elevated levels of abortive transcripts, and fully labeled pre-mRNA is more stable than its uridine-only counterpart. Cell culture experiments show that a small number of alternative splicing events were modestly, but statistically significantly influenced by metabolic labeling with 4sU at concentrations considered to be tolerable (40 μM). We conclude that at high 4sU incorporation rates small, but noticeable changes in pre-mRNA splicing can be detected when splice sites deviate from consensus. Given these potential 4sU artifacts, we suggest that appropriate controls for metabolic labeling experiments need to be included in future labeling experiments.

Molecular and biological characterisation of an Indian variant of Chrysanthemum stunt viroid

Chrysanthemum stunt viroid (CSVd), is known to cause severe stunting symptoms in chrysanthemum. Here, we have identified a CSVd strain that infects chrysanthemum in India and have biologically characterised it by generating in vitro RNA transcripts from the cloned viroid genome. Transcripts inoculated on chrysanthemum caused severe stunting (characteristic of the viroid) and mottling in chrysanthemum and stunting symptoms in cucumber var. Summer green is also reported. Sequence changes in the Terminal Conserved region (TCR) corroborated with change in RNA secondary structure might have implications associated with the viroid pathogenicity. This is the first report of CSVd causing symptoms on cucumber plants.

Changes in RNA secondary structure affect NS1 protein expression during early stage influenza virus infection

RNA secondary structures play a key role in splicing, gene expression, microRNA biogenesis, RNA editing, and other biological processes. The importance of RNA structures has been demonstrated in the life cycle of RNA-containing viruses, including the influenza virus. At least two regions of conserved secondary structure in NS segment (+) RNA are predicted to vary among influenza virus strains with respect to thermodynamic stability; both fall in the NS1 open reading frame. The NS1 protein is involved in multiple virus-host interaction processes, and its main function is to inhibit the cellular immune response to viral infection. Using a reverse genetics approach, four influenza virus strains were constructed featuring mutations that have different effects on RNA secondary structure. Growth curve experiments and ELISA data show that, at least in the first viral replication cycle, mutations G123A and A132G affecting RNA structure in the (82–148) NS RNA region influence NS1 protein expression.

RNA editing by ADAR1 leads to context-dependent transcriptome-wide changes in RNA secondary structure

Adenosine deaminase acting on RNA 1 (ADAR1) is the master RNA editor, catalyzing the deamination of adenosine to inosine. RNA editing is vital for preventing abnormal activation of cytosolic nucleic acid sensing pathways by self-double-stranded RNAs. Here we determine, by parallel analysis of RNA secondary structure sequencing (PARS-seq), the global RNA secondary structure changes in ADAR1 deficient cells. Surprisingly, ADAR1 silencing resulted in a lower global double-stranded to single-stranded RNA ratio, suggesting that A-to-I editing can stabilize a large subset of imperfect RNA duplexes. The duplexes destabilized by editing are composed of vastly complementary inverted Alus found in untranslated regions of genes performing vital biological processes, including housekeeping functions and type-I interferon responses. They are predominantly cytoplasmic and generally demonstrate higher ribosomal occupancy. Our findings imply that the editing effect on RNA secondary structure is context dependent and underline the intricate regulatory role of ADAR1 on global RNA secondary structure.

Single-gene dual-color reporter cell line to analyze RNA synthesis in vivo

RNA synthesis occurs through the multi-step process of transcription which consists of initiation, elongation, termination, and cleavage of the nascent RNA. In recent years, post-initiation events have attracted considerable attention as regulatory steps in gene expression. In particular, changes in elongation rate have been proposed to alter RNA fate either through changes in RNA secondary structure or recruitment of trans-acting factors, but systematic approaches for perturbing and measuring elongation rate are currently lacking. Here, we describe a system for precisely measuring elongation dynamics for single nascent transcripts at a single gene locus in human cell lines. The system is based on observing the production of fluorescently labeled RNA stem loops which flank a region of interest. The region of interest can be altered using flp recombinases, thus allowing one to study the effects of cis-acting sequences on transcription rate. The dual-color RNAs which are made during this process are exported and translated, thus enabling visualization of each step in gene expression.

Characterizing RNA Excited States Using NMR Relaxation Dispersion.

Changes in RNA secondary structure play fundamental roles in the cellular functions of a growing number of noncoding RNAs. This chapter describes NMR-based approaches for characterizing microsecond-to-millisecond changes in RNA secondary structure that are directed toward short-lived and low-populated species often referred to as "excited states." Compared to larger scale changes in RNA secondary structure, transitions toward excited states do not require assistance from chaperones, are often orders of magnitude faster, and are localized to a small number of nearby base pairs in and around noncanonical motifs. Here, we describe a procedure for characterizing RNA excited states using off-resonance R1ρ NMR relaxation dispersion utilizing low-to-high spin-lock fields (25-3000 Hz). R1ρ NMR relaxation dispersion experiments are used to measure carbon and nitrogen chemical shifts in base and sugar moieties of the excited state. The chemical shift data are then interpreted with the aid of secondary structure prediction to infer potential excited states that feature alternative secondary structures. Candidate structures are then tested by using mutations, single-atom substitutions, or by changing physiochemical conditions, such as pH and temperature, to either stabilize or destabilize the candidate excited state. The resulting chemical shifts of the mutants or under different physiochemical conditions are then compared to those of the ground and excited states. Application is illustrated with a focus on the transactivation response element from the human immune deficiency virus type 1, which exists in dynamic equilibrium with at least two distinct excited states.

Evolutionary characterization of non-structural gene of H9N2 influenza viruses isolated from Asia during 2008–2012

The full-length non-structural (NS) gene of seven H9N2 isolates (2008–2012) was amplified and genetically analyzed. The NS gene of these isolates belongs to the Y439 sublineage and with an average of 96.4 % identity clustered into two subgroups largely based on their time of isolation. The Ka/Ks ratio was calculated at 0.38 for subgroup 1 and 0.42 for subgroup 2 indicates that purifying selection dominates the evolution of the NS H9 in Iran. The third position of codons values, ranging from 0.371 to 0.392, confirms that synonymous codon usage in NS of the H9N2 viruses was less biased. To investigate the evolution of the NS gene of H9N2 viruses isolated from Asia, phylogenetic analysis was conducted. The results showed that Y439 sublineage with various clusters circulated in Korea, the Middle East, and Pakistan, whereas G1 sublineage existed in China. Homology analysis showed that the NS gene of Korea and Israel isolates grouped into distinct clusters, while most of Iranian isolates were closely related to the Pakistan viruses. Prediction of RNA pseudoknot within NS1 gene using RNA folding path showed eight patterns with different free energy levels. Estimation of free energies suggests destabilization of the pseudoknot and changes in RNA secondary structure may be subject to evolution of NS gene in China.

Sensitive measurement of single-nucleotide polymorphism-induced changes of RNA conformation: application to disease studies

Single-nucleotide polymorphisms (SNPs) are often linked to critical phenotypes such as diseases or responses to vaccines, medications and environmental factors. However, the specific molecular mechanisms by which a causal SNP acts is usually not obvious. Changes in RNA secondary structure emerge as a possible explanation necessitating the development of methods to measure the impact of single-nucleotide variation on RNA structure. Despite the recognition of the importance of considering the changes in Boltzmann ensemble of RNA conformers in this context, a formal method to perform directly such comparison was lacking. Here, we solved this problem and designed an efficient method to compute the relative entropy between the Boltzmann ensembles of the native and a mutant structure. On the basis of this theoretical progress, we developed a software tool, remuRNA, and investigated examples of its application. Comparing the impact of common SNPs naturally occurring in populations with the impact of random point mutations, we found that structural changes introduced by common SNPs are smaller than those introduced by random point mutations. This suggests a natural selection against mutations that significantly change RNA structure and demonstrates, surprisingly, that randomly inserted point mutations provide inadequate estimation of random mutations effects. Subsequently, we applied remuRNA to determine which of the disease-associated non-coding SNPs are potentially related to RNA structural changes.

Evolution of Hox Post-Transcriptional Regulation by Alternative Polyadenylation and MicroRNA Modulation Within 12 Drosophila Genomes

Hox genes encode a family of transcriptional regulators that operate differential developmental programs along the anteroposterior axis of bilateral animals. Regulatory changes affecting Hox gene expression are believed to have been crucial for the evolution of animal body plans. In Drosophila melanogaster, Hox expression is post-transcriptionally regulated by microRNAs (miRNAs) acting on target sites located in the 3' untranslated regions (3'UTRs) of Hox mRNAs. Notably, recent work has shown that during D. melanogaster development Hox genes produce mRNAs with variable 3'UTRs (short and long forms) in different sets of tissues as a result of alternative polyadenylation; importantly, Hox short and long 3'UTRs contain very different target sites for miRNAs. Here, we use a computational approach to explore the evolution of Hox 3'UTRs treated with especial regard to miRNA regulation. Our work is focused on the 12 Drosophila species for which genomic sequences are available and shows, first, that alternative polyadenylation of Hox transcripts is a feature shared by all drosophilids tested in the study. Second, that the regulatory impact of miRNAs is evolving very fast within the Drosophila group. Third, that in contrast to the low degree of primary sequence conservation, Hox 3'UTR regions within the group show very similar RNA topology indicating that RNA structure is under strong selective pressure. Finally, we also demonstrate that Hox alternative polyadenylation can remodel the control regions seen by miRNAs by at least two mechanisms: via adding new cis-regulatory sequences-in the form of miRNA target sites-to short 3'UTR forms as well as by modifying the regulatory impact of miRNA target sites in short 3'UTR forms through changes in RNA secondary structure caused by the use of distal polyadenylation signals.

How do bacteria sense and respond to low temperature?

Rigidification of the membrane appears to be the primary signal perceived by a bacterium when exposed to low temperature. The perception and transduction of the signal then occurs through a two-component signal transduction pathway consisting of a membrane-associated sensor and a cytoplasmic response regulator and as a consequence a set of cold-regulated genes are activated. In addition, changes in DNA topology due to change in temperature may also trigger cold-responsive mechanisms. Inducible proteins thus accumulated repair the damage caused by cold stress. For example, the fluidity of the rigidified membrane is restored by altering the levels of saturated and unsaturated fatty acids, by altering the fatty acid chain length, by changing the proportion of cis to trans fatty acids and by changing the proportion of anteiso to iso fatty acids. Bacteria could also achieve membrane fluidity changes by altering the protein content of the membrane and by altering the levels of the type of carotenoids synthesized. Changes in RNA secondary structure, changes in translation and alteration in protein conformation could also act as temperature sensors. This review highlights the various strategies by which bacteria senses low temperature signal and as to how it responds to the change.

Genetic characterization of orthobunyavirus Melao, strains BE AR633512 and BE AR8033, and experimental infection in golden hamsters (Mesocricetus auratus)

Melao virus (MELV) strains BE AR8033 and BE AR633512 were isolated from pools of Ochlerotatus scapularis mosquitoes in Belém, Pará State (1955), and Alta Floresta, Rondônia State (2000), Brazil, respectively. The aim of the present study was to molecularly characterize these strains and to describe the histopathological, biochemical and immunological changes in golden hamsters (Mesocricetus auratus) following intraperitoneal injection of MELV strains. Hamsters were susceptible to both of the MELV strains studied. Viraemia was observed 3-6 days post-infection (p.i.) for BE AR633512 and only on the second day p.i. for BE AR8033. Neutralizing antibodies against both strains were detected in blood samples obtained at 5 days p.i. and persisted up to 30 days p.i. Aspartate aminotransferase, alanine aminotransferase and blood urea nitrogen were significantly altered in animals infected with the two MELV strains, while creatinine was only altered in animals inoculated with BE AR633512. Histopathological changes were observed in the central nervous system, liver, kidney and spleen of hamsters, and infection was confirmed by detection of specific MELV antigens by immunohistochemistry. Strain BE AR633512 caused more severe tissue damage than strain BE AR8033, showing increased neurovirulence and pathogenicity. Genetic analysis based on the full-length sequences of the glycoprotein (Gn and Gc) and nucleocapsid protein (N) genes revealed high levels of homology between the MELV strains. Interestingly, the greatest genetic divergence was found for the Gn gene of strain BE AR633512, in which several synonymous and non-synonymous mutations causing changes in RNA secondary structure were observed. Further studies will be necessary to investigate the role of Gn and Gc mutations in the MELV pathogenicity.

An RNA conformational shift in recent H5N1 influenza A viruses

Recent outbreaks of avian influenza are being caused by unusually virulent H5N1 strains. It is unknown what makes these recent H5N1 strains more aggressive than previously circulating strains. Here, we have compared more than 3000 RNA sequences of segment 8 of type A influenza viruses and found a unique single nucleotide substitution typically associated with recent H5N1 strains. By phylogenetic analysis, biochemical and biophysical experiments, we demonstrate that this substitution dramatically affects the equilibrium between a hairpin and a pseudoknot conformation near the 3' splice-site of the NS gene. This conformational shift may have consequences for splicing regulation of segment 8 mRNA. Our data suggest that besides changes at the protein level, changes in RNA secondary structure should be seriously considered when attempting to explain influenza virus evolution. Supplementary data are available at Bioinformatics online.

RNA Secondary Structure Switching during DNA Synthesis Catalyzed by HIV-1 Reverse Transcriptase

Changes in RNA secondary structure have been found to play important roles in translational regulation, protein synthesis, and mRNA splicing. In studies utilizing a 66 nucleotide RNA template with a stable hairpin structure, we have examined the effects of RNA secondary structure on HIV-1 reverse transcriptase activity. We identify several pause sites in the stem of the hairpin and show that these pause sites are correlated with the free energy of melting the next base pair in the stem. We also identify a pause site appearing in the loop of the hairpin and show that this is due to the rapid formation of a new hairpin structure occurring during the progress of DNA polymerization through the hairpin. The rapid change in RNA secondary structure to form the new hairpin selectively destabilizes the major hairpin and thereby accelerates the rate at which reverse transcriptase reads through RNA secondary structure.

Mouse erythroid cells express multiple putative RNA helicase genes exhibiting high sequence conservation from yeast to mammals

RNA secondary structure is a critical determinant of RNA function in ribosome assembly, pre-mRNA splicing, mRNA translation and RNA stability. The ‘DEAD/H’ family of putative RNA helicases may help regulate these processes by utilizing intrinsic RNA-dependent ATPase activity to catalyze conformational changes in RNA secondary structure. To investigate the repertoire of DEAD/H box proteins expressed in mammals, we used PCR techniques to clone from mouse erythroleukemia (MEL) cells three new DEAD box cDNAs with high similarity to known yeast ( Saccharomyces cerevisiae) genes. mDEAD2 and mDEAD3 (mouse DEAD box proteins) are >95% identical to mouse PL10 but exhibit differential tissue-specific expression patterns; mDEAD2 and mDEAD3 are also approx. 70% identical (at the aa level) to yeast DED1 and DBP1 proteins. Members of this DEAD box subclass contain C-terminal domains with high content of Arg, Ser, Gly and Phe, reminiscent of the RS domain in several Drosophila and mammalian splicing factors. mDEAD5 belongs to a second class related to translation initiation factors from yeast (TIF1/TIF2) and mammals (eIF-4A); this class contains a novel conserved peptide motif not found in other DEAD box proteins. Northern blotting shows that mDEAD5 is differentially expressed in testis vs. somatic tissues. Thus, mouse erythroid cells produce two highly conserved families of putative RNA helicases likely to play important roles in RNA metabolism and gene expression.

Defining the binding site of Xenopus transcription factor IIIA on 5S RNA using truncated and chimeric 5S RNA molecules.

The interaction of TFIIIA with deletion fragments of Xenopus 5S RNA has been quantified using a nitrocellulose filter binding assay. TFIIIA binding was found to be more sensitive to the deletion of nucleotides from the 5' terminus of the 5S RNA as opposed to the 3' terminus. These effects have been correlated to the changes in RNA secondary structure resulting from the deletions. Nucleotides 11-108 of the intact 5S RNA provide the necessary sequence and conformational information required for the binding of TFIIIA. Synthetic 5S RNA genes have been constructed so that in vitro transcription with T7 RNA polymerase yields mature 5S RNA. The transcription factor has a higher affinity for somatic vs. oocyte 5S RNA, similar to the differential affinity of TFIIIA for the two genes. Binding studies with chimeric 5S RNA molecules indicated that the increased binding strength of somatic 5S RNA is conferred by nucleotide substitutions in the 5' half of the molecule.

Raman spectral studies of nucleic acids: III. Laser-excited spectra of ribosomal RNA

Laser-excited Raman spectra of rRNA from Escherichia coli have been obtained in the frequency range 2000-200 cm −1 for both H 2O and 2H 2O solutions. Raman lines of the phosphodiester group and of paired and unpaired purine and pyrimidine bases are readily detected. Several lines in the spectra are sensitive to changes in RNA secondary structure caused by increasing the ionic strength of solutions. The results suggest that the Raman effect will be more advantageous than infrared spectroscopy for the study of secondary structures and intermolecular interactions of nucleic acids in aqueous solution.

The optical rotatory dispersion of MS2 bacteriophage

The optical rotatory dispersions of MS2 bacteriophage, its constituent RNA 1 1 Abbreviations used: RNA, ribonucleic acid; ORD, optical rotatory dispersion; OD, optical density; PGA, poly- l-glutamic acid; CD, circular dichroism. , and coat protein subunit are described, revealing little change in RNA secondary structure upon assembly. It is concluded that the unusual MS2 protein subunit rotatory dispersion probably reflects tertiary rather than secondary structure, and that this structure persists upon assembly. Analysis of heated bacteriophage revealed an increase in protein levorotation concomitant with splitting of the particles into degraded nucleic acid and protein shells.