Double-stranded RNA induces mRNA degradation in Trypanosoma brucei

Double-stranded RNA (dsRNA) recently has been shown to give rise to genetic interference in Caenorhabditis elegans and also is likely to be the basis for phenotypic cosuppression in plants in certain instances. While constructing a plasmid vector for transfection of trypanosome cells, we serendipitously discovered that in vivo expression of dsRNA of the α-tubulin mRNA 5′ untranslated region (5′ UTR) led to multinucleated cells with striking morphological alterations and a specific block of cytokinesis. Transfection of synthetic α-tubulin 5′ UTR dsRNA, but not of either strand individually, caused the same phenotype. On dsRNA transfection, tubulin mRNA, but not the corresponding pre-mRNA, was rapidly and specifically degraded, leading to a deficit of α-tubulin synthesis. The transfected cells were no longer capable of carrying out cytokinesis and eventually died. Analysis of cytoskeletal structures from these trypanosomes revealed defects in the microtubules of the flagellar axoneme and of the flagellar attachment zone, a complex cortical structure that we propose is essential for establishing the path of the cleavage furrow at cytokinesis. Last, dsRNA-mediated mRNA degradation is not restricted to α-tubulin mRNA but can be applied to other cellular mRNAs, thus establishing a powerful tool to genetically manipulate these important protozoan parasites.

High efficiency mutagenesis, repair, and engineering of chromosomal DNA using single-stranded oligonucleotides

Homologous DNA recombination is a fundamental, regenerative process within living organisms. However, in most organisms, homologous recombination is a rare event, requiring a complex set of reactions and extensive homology. We demonstrate in this paper that Beta protein of phage λ generates recombinants in chromosomal DNA by using synthetic single-stranded DNAs (ssDNA) as short as 30 bases long. This ssDNA recombination can be used to mutagenize or repair the chromosome with efficiencies that generate up to 6% recombinants among treated cells. Mechanistically, it appears that Beta protein, a Rad52-like protein, binds and anneals the ssDNA donor to a complementary single-strand near the DNA replication fork to generate the recombinant. This type of homologous recombination with ssDNA provides new avenues for studying and modifying genomes ranging from bacterial pathogens to eukaryotes. Beta protein and ssDNA may prove generally applicable for repairing DNA in many organisms.

Organization and expression of the double-stranded RNA genome of Helminthosporium victoriae 190S virus, a totivirus infecting a plant pathogenic filamentous fungus.

The complete nucleotide sequence, 5178 bp, of the totivirus Helminthosporium vicotoriae 190S virus (Hv190SV) double-stranded RNA, was determined. Computer-assisted sequence analysis revealed the presence of two large overlapping ORFs; the 5'-proximal large ORF (ORF1) codes for the coat protein (CP) with a predicted molecular mass of 81 kDa, and the 3'-proximal ORF (ORF2), which is in the -1 frame relative to ORF1, codes for an RNA-dependent RNA polymerase (RDRP). Unlike many other totiviruses, the overlap region between ORF1 and ORF2 lacks known structural information required for translational frameshifting. Using an antiserum to a C-terminal fragment of the RDRP, the product of ORF2 was identified as a minor virion-associated polypeptide of estimated molecular mass of 92 kDa. No CP-RDRP fusion protein with calculated molecular mass of 165 kDa was detected. The predicted start codon of the RDRP ORF (2605-AUG-2607) overlaps with the stop codon (2606-UGA-2608) of the CP ORF, suggesting RDRP is expressed by an internal initiation mechanism. Hv190SV is associated with a debilitating disease of its phytopathogenic fungal host. Knowledge of its genome organization and expression will be valuable for understanding its role in pathogenesis and for potential exploitation in the development of biocontrol measures.

Synthetic transcripts of double-stranded Birnavirus genome are infectious.

We have developed a system for generation of infectious bursal disease virus (IBDV), a segmented double-stranded RNA virus of the Birnaviridae family, with the use of synthetic transcripts derived from cloned cDNA. Independent full-length cDNA clones were constructed that contained the entire coding and noncoding regions of RNA segments A and B of two distinguishable IBDV strains of serotype I. Segment A encodes all of the structural (VP2, VP4, and VP3) and nonstructural (VP5) proteins, whereas segment B encodes the RNA-dependent RNA polymerase (VP1). Synthetic RNAs of both segments were produced by in vitro transcription of linearized plasmids with T7 RNA polymerase. Transfection of Vero cells with combined plus-sense transcripts of both segments generated infectious virus as early as 36 hr after transfection. The infectivity and specificity of the recovered chimeric virus was ascertained by the appearance of cytopathic effect in chicken embryo cells, by immunofluorescence staining of infected Vero cells with rabbit anti-IBDV serum, and by nucleotide sequence analysis of the recovered virus, respectively. In addition, transfectant viruses containing genetically tagged sequences in either segment A or segment B of IBDV were generated to confirm the feasibility of this system. The development of a reverse genetics system for double-stranded RNA viruses will greatly facilitate studies of the regulation of viral gene expression, pathogenesis, and design of a new generation of live vaccines.

Molecular mechanics calculations of the riboacetal internucleotide linkage in double and triple helices.

Structures of Watson-Crick base paired 15-nucleobase oligomer strands in A-type or B-type conformation in which one strand [a strand of alternating nucleotide and riboacetal thymidine nucleoside (RT) units, RP] is DNA and the other is composed of alternating nucleotides and riboacetal nucleosides have been studied by molecular mechanics. Analogously, oligomer strands of RNA in place of DNA have been modeled. The calculations indicate that the RP strand is more stable when complexed in an A-type duplex relative to a B-type form and that this conformational preference is presumably due to the more uniform nature of the former. Nearly planar ribose rings were more commonly observed in the minimized structures of the B-type DNA.RP duplexes as compared with A-type duplexes, despite the fact that planar ribofuranose rings are known to be energetically unfavorable in oligonucleotides. Computed relative stabilities of all duplexes containing the RP strand suggest that such heteroduplexes are less stable than the corresponding double-stranded DNA and double-stranded RNA species. These findings are in agreement with experimental results which show, when equivalent sequences were compared, that a DNA.RNA control forms a more stable duplex than RP hound to a complementary single-stranded RNA strand. In contrast, molecular mechanics studies of complementary triple-helical (DNA)2.RP, (DNA)2.DNA, and (DNA)2.RNA structures indicate that the binding of RP as a Hoogsteen strand stabilizes the underlying duplex to a greater extent compared with native oligonucleotides. These calculations suggest that puckering of the ribose ring in the riboacetal linkage leads to a more favorable interaction with a complementary nucleic acid target than the proposed planar geometry and that this puckering may account for the enhanced binding of RP to a double-stranded target.

Cold shock induces a major ribosomal-associated protein that unwinds double-stranded RNA in Escherichia coli.

A 70-kDa protein was specifically induced in Escherichia coli when the culture temperature was shifted from 37 to 15 degrees C. The protein was identified to be the product of the deaD gene (reassigned csdA) encoding a DEAD-box protein. Furthermore, after the shift from 37 to 15 degrees C, CsdA was exclusively localized in the ribosomal fraction and became a major ribosomal-associated protein in cells grown at 15 degrees C. The csdA deletion significantly impaired cell growth and the synthesis of a number of proteins, specifically the derepression of heat-shock proteins, at low temperature. Purified CsdA was found to unwind double-stranded RNA in the absence of ATP. Therefore, the requirement for CsdA in derepression of heat-shock protein synthesis is a cold shock-induced function possibly mediated by destabilization of secondary structures previously identified in the rpoH mRNA.

In vitro activation of the interferon-induced, double-stranded RNA-dependent protein kinase PKR by RNA from the 3' untranslated regions of human alpha-tropomyosin.

The cellular kinase known as PKR (protein kinase RNA-activated) is induced by interferon and activated by RNA. PKR is known to have antiviral properties due to its role in translational control. Active PKR phosphorylates eukaryotic initiation factor 2 alpha and leads to inhibition of translation, including viral translation. PKR is also known to function as a tumor suppressor, presumably by limiting the rate of tumor-cell translation and growth. Recent research has shown that RNA from the 3' untranslated region (3'UTR) of human alpha-tropomyosin has tumor-suppressor properties in vivo [Rastinejad, F., Conboy, M. J., Rando, T. A. & Blau, H. M. (1993) Cell 75, 1107-1117]. Here we report that purified RNA from the 3'UTR of human alpha-tropomyosin can inhibit in vitro translation in a manner consistent with activation of PKR. Inhibition of translation by tropomyosin 3'UTR RNA was observed in a rabbit reticulocyte lysate system, which is known to contain endogenous PKR but was not seen in wheat germ lysate, which is not responsive to a known activator of PKR. A control RNA purified in the same manner as the 3'UTR RNA did not inhibit translation in either system. The inhibition of translation observed in reticulocyte lysates was prevented by the addition of adenovirus virus-associated RNA1 (VA RNAI), an inhibitor of PKR activation. Tropomyosin 3'UTR RNA was bound by immunoprecipitated PKR and activated the enzyme in an in vitro kinase assay. These data suggest that activation of PKR could be the mechanism by which tropomyosin 3'UTR RNA exerts its tumor-suppression activity in vivo.

Double-stranded-RNA-dependent protein kinase and TAR RNA-binding protein form homo- and heterodimers in vivo.

The yeast two-hybrid system and far-Western protein blot analysis were used to demonstrate dimerization of human double-stranded RNA (dsRNA)-dependent protein kinase (PKR) in vivo and in vitro. A catalytically inactive mutant of PKR with a single amino acid substitution (K296R) was found to dimerize in vivo, and a mutant with a deletion of the catalytic domain of PKR retained the ability to dimerize. In contrast, deletion of the two dsRNA-binding motifs in the N-terminal regulatory domain of PKR abolished dimerization. In vitro dimerization of the dsRNA-binding domain required the presence of dsRNA. These results suggest that the binding of dsRNA by PKR is necessary for dimerization. The mammalian dsRNA-binding protein TRBP, originally identified on the basis of its ability to bind the transactivation region (TAR) of human immunodeficiency virus RNA, also dimerized with itself and with PKR in the yeast assay. Taken together, these results suggest that complexes consisting of different combinations of dsRNA-binding proteins may exist in vivo. Such complexes could mediate differential effects on gene expression and control of cell growth.

Involvement of the double-stranded-RNA-dependent kinase PKR in interferon expression and interferon-mediated antiviral activity.

The signaling mechanisms responsible for the induced expression of interferon (IFN) genes by viral infection or double-stranded RNA (dsRNA) are not well understood. Here we investigate the role of the interferon-induced dsRNA-dependent protein kinase PKR in the regulation of IFN induction. Biological activities attributed to PKR include regulating protein synthesis, mediating IFN actions, and functioning as a possible tumor suppressor. Since binding of dsRNA is required for its activation, PKR has been considered as a candidate signal transducer for regulating IFN expression. To examine this role of PKR, loss-of-function phenotypes in stable transformants of promonocytic U-937 cells were achieved by two different strategies, overexpression of an antisense PKR transcript or a dominant negative PKR mutant gene. Both types of PKR-deficient cells were more permissive for viral replication than the control U-937 cells. As the result of PKR loss, they also showed impaired induction of IFN-alpha and IFN-beta genes in response to several inducers--specifically, encephalomyocarditis virus, lipopolysaccharide, and phorbol 12-myristate 13-acetate. Interestingly, while IFN-alpha induction by dsRNA was impaired in PKR-deficient cells, IFN-beta induction remained intact. Loss of PKR function also resulted in decreased antiviral activity as elicited by IFN-alpha and, to a greater extent, by IFN-gamma. These results implicate PKR in the regulation of several antiviral activities.

The interferon-inducible double-stranded RNA-activated protein kinase self-associates in vitro and in vivo.

The interferon-inducible double-stranded (ds) RNA-activated protein kinase (PKR) exhibits antiviral, anticellular, and antitumor activities. The mechanisms of its enzymatic activation by autophosphorylation and of the observed transdominant inhibitory phenotype of enzymatically inactive mutants have invoked PKR dimerization. Here we present direct evidence in support of PKR-PKR interaction. We show that radiolabeled PKR can specifically interact with matrix-bound unlabeled PKR in the absence of dsRNA. The self-association activity resides, in part, in the N-terminal region of 170 residues, which also constitutes the dsRNA-binding domain (DRBD). DRBD can bind to matrix-bound PKR or to matrix-bound DRBD. Dimerization of DRBD was directly demonstrated by chemical crosslinking. Affinity chromatography and electrophoretic mobility supershift assays demonstrated that mutants that fail to bind dsRNA can still exhibit protein-protein interaction. The PKR-PKR interaction could also be observed in a two-hybrid transcriptional activation assay in mammalian cells and consequently is likely to be an important feature of PKR activity in vivo.

Human autoantibody recognition of DNA.

Combinatorial IgG Fab phage display libraries prepared from a systemic lupus erythematosus (SLE) donor and a healthy donor were affinity selected against human placental DNA. Human monoclonal antibody Fab fragments specific for DNA were isolated from both libraries, although Fabs of the highest affinity were isolated only from the lupus library. Generally, apparent affinities of the Fabs for human placental DNA, purified double-stranded DNA, and denatured DNA were approximately equivalent. Surface plasmon resonance indicated Fab binding constants for a double-stranded oligodeoxynucleotide of 0.2-1.3 x 10(8) M-1. The higher-affinity Fabs, as ranked by binding to human placental DNA or to the oligonucleotide probe, tested positive in the Crithidia luciliae assay commonly used in the diagnosis of SLE, and interestingly the genes encoding the heavy-chain variable regions of these antibodies displayed evidence of only minimal somatic hypermutation. The heavy chains of the SLE Fabs were characterized by a predominance of basic residues toward the N terminus of complementarity-determining region 3 (CDR3). The crucial role of heavy-chain CDR3 (HCDR3) in high-affinity DNA recognition was suggested by the creation of DNA binding in an unrelated antibody by HCDR3 transplantation from SLE antibodies. We propose that high-affinity DNA-binding antibodies can arise in SLE without extensive somatic hypermutation in the variable-region genes because of the expression of inappropriate HCDR3s.

Genome stability pathways in head and neck cancers

Genomic instability underlies the transformation of host cells toward malignancy, promotes development of invasion and metastasis and shapes the response of established cancer to treatment. In this review, we discuss recent advances in our understanding of genomic stability in squamous cell carcinoma of the head and neck (HNSCC), with an emphasis on DNA repair pathways. HNSCC is characterized by distinct profiles in genome stability between similarly staged cancers that are reflected in risk, treatment response and outcomes. Defective DNA repair generates chromosomal derangement that can cause subsequent alterations in gene expression, and is a hallmark of progression toward carcinoma. Variable functionality of an increasing spectrum of repair gene polymorphisms is associated with increased cancer risk, while aetiological factors such as human papillomavirus, tobacco and alcohol induce significantly different behaviour in induced malignancy, underpinned by differences in genomic stability. Targeted inhibition of signalling receptors has proven to be a clinically-validated therapy, and protein expression of other DNA repair and signalling molecules associated with cancer behaviour could potentially provide a more refined clinical model for prognosis and treatment prediction. Development and expansion of current genomic stability models is furthering our understanding of HNSCC pathophysiology and uncovering new, promising treatment strategies. © 2013 Glenn Jenkins et al.

Colorimetric recognition of the coralyne-poly(dA) interaction using unmodified gold nanoparticle probes, and further detection of coralyne based upon this recognition system

Among the functional nucleic acids studied, adenine-rich nucleic acids have attracted attention due to their critical roles in many biological processes and self-assembly-based nanomaterials, especially deoxyribonucleic acids (abbreviated as poly(dA)). Therefore the ligands binding to poly(dA) might serve as potential therapeutic agents. Coralyne, a kind of planar alkaloid, has been firstly found that it could bind strongly to poly(dA). This work herein reports an approach for visual sensing of the coralyne-poly(dA) interaction. This method was based on the coralyne inducing poly(dA) into the homo-adenine DNA duplex and the difference in electrostatic affinity between single-stranded DNA and double-stranded DNA with gold nanoparticles (GNPs). Furthermore, we applied the recognition process of the interaction between coralyne and poly(dA) into specific coralyne detection with the assistance of certain software (such as Photoshop). A linear response from 0 to 728 nM was obtained for coralyne, and a detection limit of 91 nM was achieved.

Lamivudine salts with improved solubilities

To optimize solubility of drugs, current strategies mainly focus on engineering and screening of smart crystal phases. Two salts of the anti-human immunodeficiency virus (HIV) drug lamivudinenamely, lamivudine hydrochloride and lamivudine hydrochloride monohydrate, were prepared in the course of screening the crystallization conditions of lamivudine duplex, an uncommon DNA-mimic, double-stranded helical structure made up of partially protonated drug pairs. Here, water solubilities of lamivudine hydrochloride, lamivudine hydrochloride monohydrate, and lamivudine duplex are reported. The aqueous solubility of this anti-HIV drug was significantly increased in both salts and also in lamivudine duplex in relation to the water solubility of lamivudine form II. In comparison with the lamivudine form II incorporated into therapeutic formulations, the drug solubility was increased at a temperature of 299 +/- 2 K by factors of 1.2, 3.3, and 4.5 in lamivudine hydrochloride, lamivudine hydrochloride monohydrate, and lamivudine duplex, respectively, demonstrating that this solid-state property of lamivudine can be improved by crystal engineering strategies. Solubility profiles were understood on the basis of structural and solventsolute interaction approaches. At last, correlations between solubility and crystal structures allowed for a rational approach to understand how this physicochemical feature could be enhanced by engineering new salts of the drug. (C) 2012 Wiley Periodicals, Inc. and the American Pharmacists Association J Pharm Sci 101:21432154, 2012