RNA Hairpin Research Articles

Tim-3 fosters HCC development by enhancing TGF-β-mediated alternative activation of macrophages

ObjectiveTumour-associated macrophages (TAMs) and their alternative activation contribute greatly to the development of hepatocellular carcinoma (HCC). Tim-3 is highly expressed on macrophages and regulates macrophage functions in several conditions. However,...

Loss of non-coding RNA expression from the DLK1-DIO3 imprinted locus correlates with reduced neural differentiation potential in human embryonic stem cell lines.

IntroductionPluripotent stem cells are increasingly used to build therapeutic models, including the transplantation of neural progenitors derived from human embryonic stem cells (hESCs). Recently, long non-coding RNAs (lncRNAs), including delta-like homolog 1 gene and the type III iodothyronine deiodinase gene (DLK1-DIO3) imprinted locus-derived maternally expressed gene 3 (MEG3), were found to be expressed during neural development. The deregulation of these lncRNAs is associated with various neurological diseases. The imprinted locus DLK1-DIO3 encodes abundant non-coding RNAs (ncRNAs) that are regulated by differential methylation of the locus. We aim to study the correlation between the DLK1-DIO3-derived ncRNAs and the capacity of hESCs to differentiate into neural lineages.MethodsWe classified hESC sublines into MEG3-ON and MEG3-OFF based on the expression levels of MEG3 and its downstream microRNAs as detected by quantitative reverse transcription-polymerase chain reaction (qRT-PCR). A cDNA microarray was used to analyze the gene expression profiles of hESCs. To investigate the capacity of neural differentiation in MEG3-ON and MEG3-OFF hESCs, we performed neural lineage differentiation followed by neural lineage marker expression and neurite formation analyses via qRT-PCR and immunocytochemistry, respectively. MEG3-knockdown via small interfering RNA (siRNA) and small hairpin RNA (shRNA) was used to investigate the potential causative effect of MEG3 in regulating neural lineage-related gene expression.ResultsDLK1-DIO3-derived ncRNAs were repressed in MEG3-OFF hESCs compared with those in the MEG3-ON hESCs. The transcriptome profile indicated that many genes related to nervous system development and neural-type tumors were differentially expressed in MEG3-OFF hESCs. Three independent MEG3-knockdown assays using different siRNA and shRNA constructs consistently resulted in downregulation of some neural lineage genes. Lower expression levels of stage-specific neural lineage markers and reduced neurite formation were observed in neural lineage-like cells derived from MEG3-OFF-associated hESCs compared with those in the MEG3-ON groups at the same time points after differentiation.ConclusionsRepression of ncRNAs derived from the DLK1-DIO3 imprinted locus is associated with reduced neural lineage differentiation potential in hESCs.Electronic supplementary materialThe online version of this article (doi:10.1186/scrt535) contains supplementary material, which is available to authorized users.

Molecular Simulations Studies of RNA Tetraloop Hyperstability: The Effect of Stem Length on Folding Dynamics

RNA hairpins, formed by looping oligonucleotides, are among the most common RNA secondary structures and involve conserved interactions that drive hierarchical folding and stabilize folded states. Recent modifications to all-atom molecular dynamics force fields have provided the means to quantitatively assess RNA hairpin folding and stability for comparison with experiment. This study focuses on using these techniques to analyze two hyperstable RNA hairpins, each containing a hyperstable tetraloop sequence (rGCAA) and a G-C stem of two or four basepairs. The systems were simulated for a total of 300 microseconds using replica exchange molecular dynamics, and the resulting structure ensembles were analyzed for common folded states, intra-loop hydrogen bonds, and stem base-pairing retention. We find that the longer stem produces kinetic traps with non-native loop conformations, while offering credence to supposed alternative tetraloop folds from experiment. Shorter stems produce more variable folding behaviors and demonstrate reversible folding/unfolding actions. These results offer an unbiased, thermodynamic characterization of RNA tetraloops, highlight the presence of kinetic traps, and provide critical information for kinetic folding studies of tetraloops as well as additional folding studies of larger RNA molecules.

New Insight on the Role of Plasminogen Receptor in Cancer Progression.

OBJECTIVEPlasminogen system plays a crucial role in physiological and pathological events related to tissue regeneration, wound healing, immune response, angiogenesis, invasion and metastasis. It gets activated when plasminogen associates with its cell surface receptors. Latest information on some of the well-explored plasminogen receptors such as annexin II–S100A10, cytokeratin 8, α-enolase, plasminogen receptor (KT) (Plg-R(KT)) and histone H2B has been discussed in the present review. These receptors can pave the way for effective new therapeutic and diagnostic strategies to counteract malignant diseases.CONCLUSIONThe present review concludes the key role of plasminogen receptors in extracellular matrix degradation, infiltration into surrounding tissues, neovascularization, invasion, metastasis and drug resistance. This review also discusses the possible effect of blocking these plasminogen receptors with monoclonal antibodies and DNA-based vaccination or silencing plasminogen receptor gene using small interfering RNA or short hairpin RNA to counteract cancer invasion and metastasis.

Dual-Beam Fluorescence Correlation Spectroscopy of DNA Hairpin Folding-Unfolding Reaction at Various Temperatures and Salt Concentrations

Fluorescence Correlation Spectroscopy has been used to study the conformational dynamics of DNA hairpin molecules of various loop lengths in aqueous solution. In a recent development, we are also able to monitor the temperature dependence of the processes of interest. In our 2-beam approach, the analyte molecules flow through an electrophoresis capillary under the influence of pressure driven flow or electrophoretic flow and are probed by two spatially offset probe volumes in such a way that the molecules flow sequentially from one probe volume to the next. A pair of dye-quencher molecules attached to DNA or RNA hairpin structures give rise to fluorescence fluctuations due to folding-unfolding dynamics of the hairpins. Fluorescence fluctuations from the probe volumes are analyzed using auto-correlation and cross-correlation analysis. In this way, we are able to resolve fluorescence fluctuation time constants arising from diffusion, flow, triplet blinking, and conformational fluctuations. Using a temperature controlling stage we can fix the temperature to within 0.2 degrees in the range 5 - 95 degrees Celsius, unaffected by the flow rate of the solution in the capillary. In this presentation, we will discuss recent results from our study of DNA hairpins of different loop lengths, at different temperatures and Sodium ion concentrations. We will also discuss binding and unbinding of counterions to individual nucleotides as they flow through the capillary under the influence of an applied electric field. Emphasis will be placed on how the desired information can be extracted using our unique approach to fluorescence correlation spectroscopy.

Electrostatic Interaction Effects on the Binding of Spliceosomal U1A Protein-SL2 RNA Hairpin

The U1A protein is a component of the U1 small nuclear ribonucleoprotein particle, which forms part of the spliceosome. Since U1A protein is highly charged, electrostatic interactions have been suggested to be of importance in the association of the protein with RNA and the stability of the complex. To understand this effect, positively charged residues of the protein have been mutated in three positions (K20, K22 and K50) that do not have direct interactions with the SL2 RNA.We performed extensive molecule dynamics simulations on the mutant complexes and characterized the changes in the networks of interactions. To reveal more global variations within the mutant complexes, we also determined the changes on the subsections of the proteins-RNA (called communities) that move in concert together. These communities have been calculated based on generalized correlations to account for the collective atomic fluctuations within the protein-RNA complexes. These results are in agreement with the experimental complex dissociation binding studies. We then determined a measure based on the collective atomic fluctuations of different mutant complexes that have a linear correlation with the experimentally measured dissociation constants. This correlation can be used to further predict the effect of a mutation on the dissociation constant of the U1A-SL2 RNA complexes.

Sequence Specificity in RNA-Mediated Transcriptional Attenuation Examined by Coarse-Grained MD Simulations

In RNA-mediated transcriptional attenuation, the elongation of a nascent mRNA is halted prematurely through the binding of a non-coding, antisense RNA. This process is initiated by a kissing loop complex between the antisense and target hairpins. Complementarity then favors extended inter-strand base pairing; however, transcriptional attenuation requires rapid kinetics, which depend highly on sequence. Computational prediction of sequences that rapidly form extended complexes would aid in the rational design of mutually orthogonal regulators, allowing their simultaneous application to synthetic systems without generating crosstalk. Additionally, probing the energetics of these systems provides insight into the fundamental relationship between RNA sequence, structure and function.Here, we employ coarse-grained molecular dynamics (MD) simulations to investigate the potential of synthetic RNA hairpins, derived from prokaryotic attenuation systems such as pT181, to form extended complexes with their antisense partners. The resulting trajectories of an experimentally known RNA-attenuator system show a strong propensity for extended inter-strand base pairing and the formation of stable intermediates. In contrast, a sequentially similar, but non-functional system shows almost no potential for extended inter-strand base pairing. This observation was consistent even when the system was subjected to increased temperatures and external pulling forces that greatly increased the rate of complex formation in the known attenuator system. These results suggest that changes in the energetic landscape of inter-strand base pairing can be captured by coarse-grained MD simulations and provide a route for the prediction of new, orthogonal sequences capable of transcriptional attenuation in synthetic systems.

Specific Binding of the Nucleocapsid Protein Transforms the Folding Landscape of the HIV-1 TAR RNA Hairpin

Retroviral nucleocapsid (NC) proteins are nucleic acid chaperones that play a key role in the viral life cycle. During reverse transcription, HIV-1 NC destabilizes nucleic acids, including the transactivation response (TAR) hairpin to facilitate their structural re-arrangement into the lowest free energy conformations. By combining single molecule optical tweezers measurements with mfold-based free energy calculations of the unfolding landscape, we determine the equilibrium TAR stability and characterize the unfolding transition state in both the presence and absence of NC. Our results show that protein binding occurs specifically at guanines that border structural defects in the upper part of TAR hairpin. This results in preferential destabilization by NC of this part of TAR, thereby shifting the transition state closer to the bottom of the TAR stem, leading to a shorter critical unfolding length. Thus, NC facilitates TAR RNA annealing to its complementary DNA hairpin while having little effect on the stability of the final annealed duplexes. These results provide the first direct measurement of alterations in an RNA folding landscape that are induced by protein-RNA interactions, and are a novel case study of the effect of a protein that specifically destabilizes particular elements of the nucleic acid secondary structure.

RNA Unwinding by the Helicase Mtr4p and the TRAMP Complex Investigated via High-Resolution Optical Trapping

We present single-molecule high-resolution optical trapping measurements of RNA unwinding by the Ski2-like nuclear helicase Mtr4p both alone and within the Trf4/Air2/Mtr4 polyadenylation (TRAMP) complex. RNA helicases such as Mtr4p play key roles in RNA metabolism. Mtr4p is thought to assist RNA degradation by the exosome presumably by unwinding RNA structures. In addition, the TRAMP complex appends short 3′ poly(A) tails to RNA meant for degradation by the exosome. Mtr4p within the TRAMP complex plays a crucial role in regulating the polyadenylation activity, limiting the poly(A) tail length. The unwinding activity of Mtr4p is required and indeed within the TRAMP complex, Mtr4p unwinds duplex RNA 10x faster than when alone. However, the dynamic details of the RNA unwinding activity and interaction of Mtr4p and TRAMP and indeed the Ski2-like family of RNA helicases are unknown. We have performed high-resolution RNA hairpin unwinding experiments for both Mtr4p and TRAMP. We find that Mtr4p unwinds RNA duplexes in a single or multiple bursts with a mean step size of 9 base pairs. The unwinding is irreversible suggesting that Mtr4p remains stably bound after unwinding the duplex, preventing re-annealing. TRAMP unwinding experiments reveal a faster molecular recognition by the Mtr4p helicase when in complex as well as enhanced RNA duplex unwinding dynamics. Combined high-resolution trapping and fluorescence experiments underway may reveal how the competition within TRAMP between the 3′-to-5′ Mtr4p helicase and the 5′-to-3′ Trf4p polymerase is resolved dynamically.

Research on the Repeated Sequences among tRNA Sequences

Many theories thought that present-day tRNA sequences evolved from some short RNA hairpins which contain a simple stem-loop structure. To find out these significant fragment sequences, the repeated sequences of different length within 3420 tRNA sequences are counted and analyzed. The results show that: 1) the probability of occurrence P(i) with the given repeated sequences i follows a power-law distribution when the length K of repeated sequences is longer than four bases, and in this case, the total number N(n) of occurrence with the repeated times n follows a power-law distribution too; 2) the sequence of the length K which repeats the largest times is just only sequence of the length K-b wobbling b bases on its left or right side (b varies between 1 and K-1); 3) the same repeated sequences are found nearly at the identical site in different tRNA sequences as the length K of repeated sequences is longer than five bases. Then a hypothesis of the origin and evolution mechanisms of tRNA sequences is proposed and discussed.

Dynamic visualization of transcription and RNA subcellular localization in zebrafish.

Live imaging of transcription and RNA dynamics has been successful in cultured cells and tissues of vertebrates but is challenging to accomplish in vivo. The zebrafish offers important advantages to study these processes--optical transparency during embryogenesis, genetic tractability and rapid development. Therefore, to study transcription and RNA dynamics in an intact vertebrate organism, we have adapted the MS2 RNA-labeling system to zebrafish. By using this binary system to coexpress a fluorescent MS2 bacteriophage coat protein (MCP) and an RNA of interest tagged with multiple copies of the RNA hairpin MS2-binding site (MBS), live-cell imaging of RNA dynamics at single RNA molecule resolution has been achieved in other organisms. Here, using a Gateway-compatible MS2 labeling system, we generated stable transgenic zebrafish lines expressing MCP, validated the MBS-MCP interaction and applied the system to investigate zygotic genome activation (ZGA) and RNA localization in primordial germ cells (PGCs) in zebrafish. Although cleavage stage cells are initially transcriptionally silent, we detect transcription of MS2-tagged transcripts driven by the βactin promoter at ∼ 3-3.5 h post-fertilization, consistent with the previously reported ZGA. Furthermore, we show that MS2-tagged nanos3 3'UTR transcripts localize to PGCs, where they are diffusely cytoplasmic and within larger cytoplasmic accumulations reminiscent of those displayed by endogenous nanos3. These tools provide a new avenue for live-cell imaging of RNA molecules in an intact vertebrate. Together with new techniques for targeted genome editing, this system will be a valuable tool to tag and study the dynamics of endogenous RNAs during zebrafish developmental processes.

Identification and diagnostic performance of a small RNA within the PCA3 and BMCC1 gene locus that potentially targets mRNA.

PCA3 is a long noncoding RNA (lncRNA) with unknown function, upregulated in prostate cancer. LncRNAs may be processed into smaller active species. We hypothesized this for PCA3. We computed feasible RNA hairpins within the BMCC1 gene (encompassing PCA3) and searched a prostate transcriptome for these. We measured expression using qRT-PCR in three cohorts of prostate cancer tissues (n = 60), exfoliated urinary cells (n = 484 with cancer and n = 166 controls), and in cell lines (n = 22). We used in silico predictions and RNA knockup to identify potential mRNA targets of short transcribed RNAs. We predicted 13 hairpins, of which PCA3-shRNA2 was most abundant within the prostate transcriptome. PCA3-shRNA2 is located within intron 1 of PCA3 and appears regulated by androgens. Expression of PCA3-shRNA2 was upregulated in malignant prostatic tissues, exfoliated urinary cells from men with prostate cancer (13-273 fold change; t test P < 0.003), and closely correlated to PCA3 expression (r = 0.84-0.93; P < 0.001). Urinary PCA3-shRNA2 (C-index, 0.75-0.81) and PCA3 (C-index, 0.78) could predict the presence of cancer in most men. PCA3-shRNA2 knockup altered the expression of predicted target mRNAs, including COPS2, SOX11, WDR48, TEAD1, and Noggin. PCA3-shRNA2 expression was negatively correlated with COPS2 in patient samples (r = -0.32; P < 0.001). We identified a short RNA within PCA3, whose expression is correlated to PCA3, which may target mRNAs implicated in prostate biology. This short RNA is stable ex vivo, suggesting a role as a robust biomarker. We identify cytoplasmic enrichment of this RNA and potential targeting of mRNAs implicated in prostate carcinogenesis.

Energy landscapes, folding mechanisms, and kinetics of RNA tetraloop hairpins.

RNA hairpins play a pivotal role in a diverse range of cellular functions, and are integral components of ribozymes, mRNA, and riboswitches. However, the mechanistic and kinetic details of RNA hairpin folding, which are key determinants of most of its biological functions, are poorly understood. In this work, we use the discrete path sampling (DPS) approach to explore the energy landscapes of two RNA tetraloop hairpins, and provide insights into their folding mechanisms and kinetics in atomistic detail. Our results show that the potential energy landscapes have a distinct funnel-like bias toward the folded hairpin state, consistent with efficient structure-seeking properties. Mechanistic and kinetic information is analyzed in terms of kinetic transition networks. We find microsecond folding times, consistent with temperature jump experiments, for hairpin folding initiated from relatively compact unfolded states. This process is essentially driven by an initial collapse, followed by rapid zippering of the helix stem in the final phase. Much lower folding rates are predicted when the folding is initiated from extended chains, which undergo longer excursions on the energy landscape before nucleation events can occur. Our work therefore explains recent experiments and coarse-grained simulations, where the folding kinetics exhibit precisely this dependency on the initial conditions.

Glioma-derived versican promotes tumor expansion via glioma-associated microglial/macrophages Toll-like receptor 2 signaling.

Accumulation and infiltration of microglia/brain macrophages around and into glioma tissue promote tumor invasion and expansion. One tumor-promoting mechanism of microglia/brain macrophages is upregulation of membrane type 1 matrix metalloprotease (MT1-MMP), which promotes the degradation of extracellular matrix. MT1-MMP upregulation is induced by soluble factors released by glioma cells activating microglial Toll-like receptor 2 (TLR2). Versican identified by proteomics was silenced in glioma cells by short interference RNA and short hairpin RNA approaches and studied in vitro and after injection into mouse brains or organotypic brain slices. The splice variants V0/V1 of the endogenous TLR2 ligand versican are highly expressed in mouse and human glioma tissue. Versican-silenced gliomas induced less MT1-MMP expression in microglia both in vitro and in vivo, which resulted in smaller tumors and longer survival rates as compared with controls. Recombinant versican V1 induced significantly higher levels of MT1-MMP in wild-type microglia compared with untreated and treated TLR2 knockout microglial cells. Using glioma-injected organotypic brain slices, we found that the impact of versican signaling on glioma growth depended on the presence of microglia. Moreover, we found that TLR2 expression is upregulated in glioma-associated microglia but not in astrocytes. Additionally, an established TLR2 neutralizing antibody reduced glioma-induced microglial MT1-MMP expression as well as glioma growth ex vivo. Our results show that versican released from glioma promotes tumor expansion through glioma-associated microglial/macrophage TLR2 signaling and subsequent expression of MT1-MMP. This signaling cascade might be a novel target for glioma therapies.

Synergetic regulation of translational reading-frame switch by ligand-responsive RNAs in mammalian cells.

Distinct translational initiation mechanisms between prokaryotes and eukaryotes limit the exploitation of prokaryotic riboswitch repertoire for regulatory RNA circuit construction in mammalian application. Here, we explored programmed ribosomal frameshifting (PRF) as the regulatory gene expression platform for engineered ligand-responsive RNA devices in higher eukaryotes. Regulation was enabled by designed ligand-dependent conformational rearrangements of the two cis-acting RNA motifs of opposite activity in -1 PRF. Particularly, RNA elements responsive to trans-acting ligands can be tailored to modify co-translational RNA refolding dynamics of a hairpin upstream of frameshifting site to achieve reversible and adjustable -1 PRF attenuating activity. Combined with a ligand-responsive stimulator, synthetic RNA devices for synergetic translational-elongation control of gene expression can be constructed. Due to the similarity between co-transcriptional RNA hairpin folding and co-translational RNA hairpin refolding, the RNA-responsive ligand repertoire provided in prokaryotic systems thus becomes accessible to gene-regulatory circuit construction for synthetic biology application in mammalian cells.

Sustained translational repression of lactate dehydrogenase 1 in Toxoplasmagondii bradyzoites is conferred by a small regulatory RNA hairpin.

In response to environmental stresses, Toxoplasmagondii induces a global translational repression which allows for the remodeling of its transcriptome. While some transcripts are preferentially translated, another subset is translationally repressed and maintained in bradyzoites. Although little is known of how transcripts are targeted for sustained translational repression, the targeting probably operates through an RNA-centric mechanism relying on the recognition of cis-acting elements. In this study, we sought to determine if the targeting of transcripts through recognizable cis-acting elements could be responsible for the transcript-specific sustained translational repression displayed by Toxoplasma bradyzoites. We examined the UTRs of a translationally repressed gene, lactate dehydrogenase 1, and found a 40 nucleotide regulatory element in its 5'UTR. This element specifically induces translational repression in otherwise constitutively expressed transcripts. Mutational studies revealed that the formation of a small 16 nucleotide regulatory RNA hairpin is essential for this activity. We suggest that this hairpin may act as the nucleation site for the binding of an as yet to be identified trans-acting factor that allows for the transcript to be targeted for translational repression removal from the active translational pool. To our knowledge, this is the first report characterizing a specific cis-acting element contributing to post-transcriptional gene regulation in Toxoplasma and suggests the presence of a pathway by which the parasites can recognize, identify and specifically target transcripts for sustained translational repression under stressful conditions.

STUDY OF THE INITIAL STEPS OF POTATO VIRUS X ASSEMBLY

Potato virus X (Potexvirus) is an important pathogen of number of economically significant agricultural plants of the Solanaceae family. Potato virus X (PVX) virions are flexible filamentous particles 515 nm in length and 13.5 nm in diameter with a helical structure. PVX genome consists of a single-stranded RNA (6435 nucleotides) which is capped and polyadenylated. The study of various stages of the infection process of plant viruses during infection, including the assembly of viral particles is of great practical interest. Identification of these mechanisms can be the basis for developing new approaches in virus-free crop production. Herein, the initial stages of potato virus X (PVX) virion assembly were examined on the example of virus-like particles (VLPs) produced by incubation of PVX RNA with PVX coat protein (CP) in vitro. The formation of identical set of VLP with discrete size under different incubation conditions (ionic strength, pH) was shown. However, the amount of VLP of a different size changes depending on incubation conditions. Most efficient VLP similar to the native virion size assembly has been achieved in a buffer of low ionic strength at pH 8.5. PVX RNA fragments from 800 to 5700 nt in length which were protected by coat protein within VLPs were isolated. Their individual analysis confirmed that all of them represent the 5´-terminal fragments of the genomic PVX RNA of different lengths. Thus, it was revealed that RNAs within VLP are genomic RNA 5´-terminal fragments of different lengths. PVX virions assembly initiation at the RNA 5´-end which is cooperatively extending in the 5´- to 3´-end direction was confirmed. Nucleotide sequence analysis of RNA fragments isolated from VLP of different sizes showed that sites capable to form RNA hairpins were discovered near the RNA 3´-end. They could act as «stop-signals» that prevent CP and RNA interaction and continuous cooperative assembling PVX VLP and virions. Probably, CP could «melt» RNA hairpins more or less efficiently and overcome «stop-signals» during the viral particles formation, depending on the conditions of incubation with the RNA in vitro. Eeywords: plant viruses, potato virus X, virus-like particles, RNA, coat protein, virion assembly. The study of various stages of the infection process of plant viruses including the assembly of viral particles is of great practical interest. Identifica- tion of these mechanisms can be the basis for developing new approaches in virus-free crop production. Potato virus O (PVX), a typical Potexvirus (Alphaflex- iviridae), is a common pathogen of many plant species of Solanaceae family, including number of economically significant agricultural plants, particularly potato plants. It should be noted that potato, together with rice, wheat and corn, are the main food stuffs in the world. PVX virions are flexible filamentous particles 515 nm in length and 13.5 nm in diameter with a helical structure. PVX genome consists of a single- stranded RNA (6435 nucleotides) which is capped and polyadenylated. About 1300 subunits of viral coat protein (CP) are polymerized to form polar helix with a pitch of 3.6 nm, each coil consist of 8-9 CP subunits, with viral RNA being encapsulated into the CP helix (1). PVX is the first filamentous plant virus reconstructed in vitro from RNA and CP (2). No differences between native virions and the reconstructed parti-

ИЗУЧЕНИЕ ПЕРВЫХ СТАДИЙ СБОРКИ ВИРИОНА У Х-ВИРУСА КАРТОФЕЛЯ

Potato virus X ( Potexvirus ) is an important pathogen of number of economically significant agricultural plants of the Solanaceae family. Potato virus X (PVX) virions are flexible filamentous particles 515 nm in length and 13.5 nm in diameter with a helical structure. PVX genome consists of a single-stranded RNA (6435 nucleotides) which is capped and polyadenylated. The study of various stages of the infection process of plant viruses during infection, including the assembly of viral particles is of great practical interest. Identification of these mechanisms can be the basis for developing new approaches in virus-free crop production. Herein, the initial stages of potato virus X (PVX) virion assembly were examined on the example of virus-like particles (VLPs) produced by incubation of PVX RNA with PVX coat protein (CP) in vitro. The formation of identical set of VLP with discrete size under different incubation conditions (ionic strength, pH) was shown. However, the amount of VLP of a different size changes depending on incubation conditions. Most efficient VLP similar to the native virion size assembly has been achieved in a buffer of low ionic strength at pH 8.5. PVX RNA fragments from 800 to 5700 nt in length which were protected by coat protein within VLPs were isolated. Their individual analysis confirmed that all of them represent the 5´-terminal fragments of the genomic PVX RNA of different lengths. Thus, it was revealed that RNAs within VLP are genomic RNA 5´-terminal fragments of different lengths. PVX virions assembly initiation at the RNA 5´-end which is cooperatively extending in the 5´to 3´-end direction was confirmed. Nucleotide sequence analysis of RNA fragments isolated from VLP of different sizes showed that sites capable to form RNA hairpins were discovered near the RNA 3´-end. They could act as «stop-signals» that prevent CP and RNA interaction and continuous cooperative assembling PVX VLP and virions. Probably, CP could «melt» RNA hairpins more or less efficiently and overcome «stop-signals» during the viral particles formation, depending on the conditions of incubation with the RNA in vitro.

A coarse-grained model with implicit salt for RNAs: predicting 3D structure, stability and salt effect.

To bridge the gap between the sequences and 3-dimensional (3D) structures of RNAs, some computational models have been proposed for predicting RNA 3D structures. However, the existed models seldom consider the conditions departing from the room/body temperature and high salt (1M NaCl), and thus generally hardly predict the thermodynamics and salt effect. In this study, we propose a coarse-grained model with implicit salt for RNAs to predict 3D structures, stability, and salt effect. Combined with Monte Carlo simulated annealing algorithm and a coarse-grained force field, the model folds 46 tested RNAs (≤45 nt) including pseudoknots into their native-like structures from their sequences, with an overall mean RMSD of 3.5 Å and an overall minimum RMSD of 1.9 Å from the experimental structures. For 30 RNA hairpins, the present model also gives the reliable predictions for the stability and salt effect with the mean deviation ∼ 1.0 °C of melting temperatures, as compared with the extensive experimental data. In addition, the model could provide the ensemble of possible 3D structures for a short RNA at a given temperature/salt condition.

RNA interference-based therapeutics: molecular platforms for infectious diseases.

The potential uses and therapeutic benefits of RNA interference (RNAi) are enormous. Recent insights into RNAi technologies have highlighted their role in analyzing the functions and regulation of gene expression in eukaryotes and further utilizing this information for identification and amelioration of many diseases. These studies have also established the role of RNAi mediated post-transcriptional gene silencing (PTGS) mechanism in mammals by several endogenous, gene regulation systems including small interfering RNAs (siRNA), micro RNA (miRNA) and small hairpin RNAs (shRNA). Moreover, these RNAi-based therapeutics have demonstrated the capability to silence therapeutically relevant genes in various in vivo models of cancer, infections autoimmune diseases and other genetic disorders. Over the past few decades, infectious diseases have been one of the leading causes of death around the world. Ubiquitously, intracellular obligate or facultative microorganisms cause serious or fatal infections and associated diseases in humans. Currently available literature suggests that infections caused by intracellular pathogens present an intriguing area, wherein RNAi technology may be effectively employed to neutralize the harmful effects of various intracellular pathogens. In this manuscript, we have emphasized on the challenges and opportunities involved in the therapy of such intracellular infections, especially employing RNAi-based interventions. We have focused our discussion on the current state-of-the-art RNAi-based therapies, which have been explored for various intracellular infections mediated by bacteria, fungi, viruses and protozoa. Nanocarrier mediated delivery of siRNA and shRNA molecules have also been found to overcome the various delivery challenges of these biotherapeutics; these have also been briefly summarized here. Furthermore, the outcomes and progresses that have been made in pre-clinical models and clinical trials have also been presented to review the numerous challenges encountered so far. Finally, we have also addressed the various future perspectives that could overcome these challenges and accelerate the progress and commercial success of these systems.