Impact of double-stranded RNA characteristics on the activation of human 2'-5'-oligoadenylate synthetase 2 (OAS2).

Human 2'-5' oligoadenylate synthetases (OAS) are a family of interferon-inducible proteins that, upon activation by double-stranded RNA, polymerize ATP into 2'-5' linked oligoadenylates. In this study, we probed the RNA cofactor specificity of the two smallest isozymes, OAS1 and OAS2. First, we developed a strategy for the expression and purification of recombinant human OAS2 from eukaryotic cells and quantified the activity of the enzyme relative to OAS1 in vitro. We then confirmed that both OAS2 domains, as opposed to only the domain containing the canonical catalytic aspartic acid triad, are required for enzymatic activity. Enzyme kinetics of both OAS1 and OAS2 in the presence of a variety of RNA binding partners enabled characterization of the maximum reaction velocity and apparent RNA-protein affinity of activating RNAs. While in this study OAS1 can be catalytically activated by dsRNA of any length greater than 19 bp, OAS2 showed a marked increase in activity with increasing dsRNA length with a minimum requirement of 35 bp. Interestingly, activation of OAS2 was also more efficient when the dsRNA contained 3'-overhangs, despite no significant impact on binding affinity. Highly structured viral RNAs that are established OAS1 activators were not able to activate OAS2 enzymatic activity based on the lack of extended stretches of dsRNA of greater than 35 bp. Together these results may highlight distinct subsets of biological RNAs to which different human OAS isozymes respond.

RNA Helicase Associated with AU-rich Element (RHAU/DHX36) Interacts with the 3'-Tail of the Long Non-coding RNA BC200 (BCYRN1).

RNA helicase associated with AU-rich element (RHAU) is an ATP-dependent RNA helicase that demonstrates high affinity for quadruplex structures in DNA and RNA. To elucidate the significance of these quadruplex-RHAU interactions, we have performed RNA co-immunoprecipitation screens to identify novel RNAs bound to RHAU and characterize their function. In the course of this study, we have identified the non-coding RNA BC200 (BCYRN1) as specifically enriched upon RHAU immunoprecipitation. Although BC200 does not adopt a quadruplex structure and does not bind the quadruplex-interacting motif of RHAU, it has direct affinity for RHAU in vitro. Specifically designed BC200 truncations and RNase footprinting assays demonstrate that RHAU binds to an adenosine-rich region near the 3'-end of the RNA. RHAU truncations support binding that is dependent upon a region within the C terminus and is specific to RHAU isoform 1. Tests performed to assess whether BC200 interferes with RHAU helicase activity have demonstrated the ability of BC200 to act as an acceptor of unwound quadruplexes via a cytosine-rich region near the 3'-end of the RNA. Furthermore, an interaction between BC200 and the quadruplex-containing telomerase RNA was confirmed by pull-down assays of the endogenous RNAs. This leads to the possibility that RHAU may direct BC200 to bind and exert regulatory functions at quadruplex-containing RNA or DNA sequences.

Studies on Structural, Morphological and Optical Properties of Chemically Deposited CdS(1-x)Se(x) Thin Films.

The thin films of CdS(1-x)Se(x) were successfully deposited over glass substrates by chemical bath deposition technique. Cadmium acetate, thiourea and sodium selenosulfate were used as source materials for Cd(2+), S(2-) and Se(2-) ions, while 2-mercaptoethanol was used as capping agent. The various deposition conditions such as precursor concentration, deposition temperature, pH and deposition time were optimized for the deposition of CdS(1-x)Se(x) thin films of good quality and the films were annealed at 200° and 300 °C. The structural, morphological, chemical and optical properties were examined by various characterization techniques and discussed in detail. The optical band gap of CdS(1-x)Se(x) thin film samples were estimated and found in the range from 2.11 to 1.79 eV for as-deposited and annealed thin films.

The RNA helicase RHAU (DHX36) suppresses expression of the transcription factor PITX1.

RNA Helicase associated with AU-rich element (RHAU) (DHX36) is a DEAH (Aspartic acid, Glumatic Acid, Alanine, Histidine)-box RNA helicase that can bind and unwind G4-quadruplexes in DNA and RNA. To detect novel RNA targets of RHAU, we performed an RNA co-immunoprecipitation screen and identified the PITX1 messenger RNA (mRNA) as specifically and highly enriched. PITX1 is a homeobox transcription factor with roles in both development and cancer. Primary sequence analysis identified three probable quadruplexes within the 3'-untranslated region of the PITX1 mRNA. Each of these sequences, when isolated, forms stable quadruplex structures that interact with RHAU. We provide evidence that these quadruplexes exist in the endogenous mRNA; however, we discovered that RHAU is tethered to the mRNA via an alternative non-quadruplex-forming region. RHAU knockdown by small interfering RNA results in significant increases in PITX1 protein levels with only marginal changes in mRNA, suggesting a role for RHAU in translational regulation. Involvement of components of the microRNA machinery is supported by similar and non-additive increases in PITX1 protein expression on Dicer and combined RHAU/Dicer knockdown. We also demonstrate a requirement of argonaute-2, a key RNA-induced silencing complex component, to mediate RHAU-dependent changes in PITX1 protein levels. These results demonstrate a novel role for RHAU in microRNA-mediated translational regulation at a quadruplex-containing 3'-untranslated region.

Adjuvants Based on Hybrid Antibiotics Overcome Resistance in Pseudomonas aeruginosa and Enhance Fluoroquinolone Efficacy.

The use of adjuvants that rescue antibiotics against multidrug-resistant (MDR) pathogens is a promising combination strategy for overcoming bacterial resistance. While the combination of ß-lactam antibiotics and ß-lactamase inhibitors has been successful in restoring antibacterial efficacy in MDR bacteria, the use of adjuvants to restore fluoroquinolone efficacy in MDR Gram-negative pathogens has been challenging. We describe tobramycin-ciprofloxacin hybrid adjuvants that rescue the activity of fluoroquinolone antibiotics against MDR and extremely drug-resistant Pseudomonas aeruginosa isolates in vitro and enhance fluoroquinolone efficacy in vivo. Structure-activity studies reveal that the presence of both tobramycin and ciprofloxacin, which are separated by a C12 tether, is critical for the function of the adjuvant. Mechanistic studies indicate that the antibacterial modes of ciprofloxacin are retained while the role of tobramycin is limited to destabilization of the outer membrane in the hybrid.

Characterization of the termini of the West Nile virus genome and their interactions with the small isoform of the 2' 5'-oligoadenylate synthetase family.

2' 5'-Oligoadenylate synthetases (OAS) are interferon-stimulated proteins that act in the innate immune response to viral infection. Upon binding viral double-stranded RNA, OAS enzymes produce 2'-5'-linked oligoadenylates that stimulate RNase L and ultimately slow viral propagation. Truncations/mutations in the smallest human OAS isoform, OAS1, results in susceptibility to West Nile virus (WNV). We have previously demonstrated in vitro the interaction between OAS1 and the 5'-terminal region of the WNV RNA genome. Here we report that the 3'-terminal region is also able to mediate specific interaction with and activation of OAS1. Binding and kinetic experiments identified a specific stem loop within the 3'-terminal region that is sufficient for activation of the enzyme. The solution conformation of the 3'-terminal region was determined by small angle X-ray scattering, and computational models suggest a conformationally restrained structure comprised of a helix and short stem loop. Structural investigation of the 3'-terminal region in complex with OAS1 is also presented. Finally, we show that genome cyclization by base pairing between the 5'- and 3'-terminal regions, a required step for replication, is not sufficient to protect WNV from OAS1 recognition in vitro. These data provide a physical framework for understanding recognition of the highly structured terminal regions of a flaviviral genome by an innate immune enzyme.

Recognition of viral RNA stem-loops by the tandem double-stranded RNA binding domains of PKR.

In humans, the double-stranded RNA (dsRNA)-activated protein kinase (PKR) is expressed in late stages of the innate immune response to viral infection by the interferon pathway. PKR consists of tandem dsRNA binding motifs (dsRBMs) connected via a flexible linker to a Ser/Thr kinase domain. Upon interaction with viral dsRNA, PKR is converted into a catalytically active enzyme capable of phosphorylating a number of target proteins that often results in host cell translational repression. A number of high-resolution structural studies involving individual dsRBMs from proteins other than PKR have highlighted the key features required for interaction with perfectly duplexed RNA substrates. However, viral dsRNA molecules are highly structured and often contain deviations from perfect A-form RNA helices. By use of small-angle X-ray scattering (SAXS), we present solution conformations of the tandem dsRBMs of PKR in complex with two imperfectly base-paired viral dsRNA stem-loops; HIV-1 TAR and adenovirus VA(I)-AS. Both individual components and complexes were purified by size exclusion chromatography and characterized by dynamic light scattering at multiple concentrations to ensure monodispersity. SAXS ab initio solution conformations of the individual components and RNA-protein complexes were determined and highlight the potential of PKR to interact with both stem and loop regions of the RNA. Excellent agreement between experimental and model-based hydrodynamic parameter determination heightens our confidence in the obtained models. Taken together, these data support and provide a framework for the existing biochemical data regarding the tolerance of imperfectly base-paired viral dsRNA by PKR.

Solution conformation of adenovirus virus associated RNA-I and its interaction with PKR.

Adenovirus virus-associated RNA (VAI) provides protection against the host antiviral response in part by inhibiting the interferon-induced double stranded RNA-activated protein kinase (PKR). VAI consists of three base-paired regions; the apical stem responsible for the interaction with double-stranded RNA binding motifs (dsRBMs) of PKR, the central stem required for inhibition, and the terminal stem. The solution conformation of VAI and VAI lacking the terminal stem were determined using SAXS that suggested extended conformations that are in agreement with their secondary structures. Solution conformations of VAI lacking the terminal stem in complex with the dsRBMs of PKR indicated that the apical stem interacts with both dsRNA-binding motifs whereas the central stem does not. Hydrodynamic properties calculated from ab initio models were compared to experimentally determined parameters for model validation. Furthermore, SAXS envelopes were used as a constraint for the in silico modeling of tertiary structure for RNA and RNA-protein complex. Finally, full-length PKR was also studied, but concentration-dependent changes in hydrodynamic parameters prevented ab initio shape determination. Taken together, results provide an improved structural framework that further our understanding of the role VAI plays in evading host innate immune responses.

Binding of G-quadruplexes to the N-terminal recognition domain of the RNA helicase associated with AU-rich element (RHAU).

Polynucleotides containing consecutive tracts of guanines can adopt an intramolecular G-quadruplex structure where multiple planar tetrads of hydrogen-bound guanines stack on top of each other. Remodeling of G-quadruplexes impacts numerous aspects of nucleotide biology including transcriptional and translational control. RNA helicase associated with AU-rich element (RHAU), a member of the ATP-dependent DEX(H/D) family of RNA helicases, has been established as a major cellular quadruplex resolvase. RHAU contains a core helicase domain responsible for ATP binding/hydrolysis/helicase activity and is flanked on either side by N- and C-terminal extensions. The N-terminal extension is required for quadruplex recognition, and we have previously demonstrated complex formation between this domain and a quadruplex from human telomerase RNA. Here we used an integrated approach that includes small angle x-ray scattering, nuclear magnetic resonance spectroscopy, circular dichroism, and dynamic light scattering methods to demonstrate the recognition of G-quadruplexes by the N-terminal domain of RHAU. Based on our results, we conclude that (i) quadruplex from the human telomerase RNA and its DNA analog both adopt a disc shape in solution, (ii) RHAU53-105 adopts a defined and extended conformation in solution, and (iii) the N-terminal domain mediates an interaction with a guanine tetrad face of quadruplexes. Together, these data form the foundation for understanding the recognition of quadruplexes by the N-terminal domain of RHAU.

Strategies for improving antimicrobial peptide production.

Antimicrobial peptides (AMPs) found in a wide range of animal, insect, and plant species are host defense peptides forming an integral part of their innate immunity. Although the exact mode of action of some AMPs is yet to be deciphered, many exhibit membrane lytic activity or interact with intracellular targets. The ever-growing threat of antibiotic resistance has brought attention to research on AMPs to enhance their clinical use as a therapeutic alternative. AMPs have several advantages over antibiotics such as broad range of antimicrobial activities including anti-fungal, anti-viral and anti-bacterial, and have not reported to contribute to resistance development. Despite the numerous studies to develop efficient production methods for AMPs, limitations including low yield, degradation, and loss of activity persists in many recombinant approaches. In this review, we outline available approaches for AMP production and various expression systems used to achieve higher yield and quality. In addition, recent advances in recombinant strategies, suitable fusion protein partners, and other molecular engineering strategies for improved AMP production are surveyed.

Recent Advances in Genetic Tools for Acinetobacter baumannii.

Acinetobacter baumannii is classified as a top priority pathogen by the World Health Organization (WHO) because of its widespread resistance to all classes of antibiotics. This makes the need for understanding the mechanisms of resistance and virulence critical. Therefore, tools that allow genetic manipulations are vital to unravel the mechanisms of multidrug resistance (MDR) and virulence in A. baumannii. A host of current strategies are available for genetic manipulations of A. baumannii laboratory-strains, including ATCC® 17978TM and ATCC® 19606T, but depending on susceptibility profiles, these strategies may not be sufficient when targeting strains newly obtained from clinic, primarily due to the latter's high resistance to antibiotics that are commonly used for selection during genetic manipulations. This review highlights the most recent methods for genetic manipulation of A. baumannii including CRISPR based approaches, transposon mutagenesis, homologous recombination strategies, reporter systems and complementation techniques with the spotlight on those that can be applied to MDR clinical isolates.

Impact of the structural integrity of the three-way junction of adenovirus VAI RNA on PKR inhibition.

Highly structured RNA derived from viral genomes is a key cellular indicator of viral infection. In response, cells produce the interferon inducible RNA-dependent protein kinase (PKR) that, when bound to viral dsRNA, phosphorylates eukaryotic initiation factor 2α and attenuates viral protein translation. Adenovirus can evade this line of defence through transcription of a non-coding RNA, VAI, an inhibitor of PKR. VAI consists of three base-paired regions that meet at a three-way junction; an apical stem responsible for the interaction with PKR, a central stem required for inhibition, and a terminal stem. Recent studies have highlighted the potential importance of the tertiary structure of the three-way junction to PKR inhibition by enabling interaction between regions of the central and terminal stems. To further investigate the role of the three-way junction, we characterized the binding affinity and inhibitory potential of central stem mutants designed to introduce subtle alterations. These results were then correlated with small-angle X-ray scattering solution studies and computational tertiary structural models. Our results demonstrate that while mutations to the central stem have no observable effect on binding affinity to PKR, mutations that appear to disrupt the structure of the three-way junction prevent inhibition of PKR. Therefore, we propose that instead of simply sequestering PKR, a specific structural conformation of the PKR-VAI complex may be required for inhibition.

Activation of 2' 5'-oligoadenylate synthetase by stem loops at the 5'-end of the West Nile virus genome.

West Nile virus (WNV) has a positive sense RNA genome with conserved structural elements in the 5' and 3' -untranslated regions required for polyprotein production. Antiviral immunity to WNV is partially mediated through the production of a cluster of proteins known as the interferon stimulated genes (ISGs). The 2' 5'-oligoadenylate synthetases (OAS) are key ISGs that help to amplify the innate immune response. Upon interaction with viral double stranded RNA, OAS enzymes become activated and enable the host cell to restrict viral propagation. Studies have linked mutations in the OAS1 gene to increased susceptibility to WNV infection, highlighting the importance of OAS1 enzyme. Here we report that the region at the 5'-end of the WNV genome comprising both the 5'-UTR and initial coding region is capable of OAS1 activation in vitro. This region contains three RNA stem loops (SLI, SLII, and SLIII), whose relative contribution to OAS1 binding affinity and activation were investigated using electrophoretic mobility shift assays and enzyme kinetics experiments. Stem loop I, comprising nucleotides 1-73, is dispensable for maximum OAS1 activation, as a construct containing only SLII and SLIII was capable of enzymatic activation. Mutations to the RNA binding site of OAS1 confirmed the specificity of the interaction. The purity, monodispersity and homogeneity of the 5'-end (SLI/II/III) and OAS1 were evaluated using dynamic light scattering and analytical ultra-centrifugation. Solution conformations of both the 5'-end RNA of WNV and OAS1 were then elucidated using small-angle x-ray scattering. In the context of purified components in vitro, these data demonstrate the recognition of conserved secondary structural elements of the WNV genome by a member of the interferon-mediated innate immune response.

Regulation of the interferon-inducible 2'-5'-oligoadenylate synthetases by adenovirus VA(I) RNA.

Foreign double-stranded RNA (dsRNA) generated during the normal course of the viral life cycle serves as a key infection recognition element by proteins of the innate immune response. To circumvent this response, all adenoviruses synthesize at least one highly structured RNA (VA(I)), which, after processing by the RNA silencing machinery, inhibits the innate immune response via a series of interactions with specific protein partners. Surprisingly, VA(I) positively regulates the activity of the interferon-induced 2'-5'-oligoadenylate synthetase (OAS) enzymes, which typically represent a key mechanism whereby host-cell protein translation is attenuated in response to foreign dsRNA. We present data investigating the regulation of the OAS1 isoform by VA(I) derivatives and demonstrate that a processed version of VA(I) lacking the terminal stem behaves as a pseudo-inhibitor of OAS1. A combination of electrophoretic mobility shift assays, dynamic light scattering, and non-denaturing mass spectrometry was used to quantitate binding affinity and characterize OAS1:VA(I) complex stoichiometry. Enzyme assays characterized the ability of VA(I) derivatives to activate OAS1. Finally, the importance of RNA 5'-end phosphorylation state is investigated, and it emphasizes its potential importance in the activation or inhibition of OAS enzymes. Taken together, these data suggest a plausible strategy whereby the virus produces a single RNA transcript capable of inhibiting a variety of members of the innate immune response.