Annexin A2 Binds RNA and Reduces the Frameshifting Efficiency of Infectious Bronchitis Virus

Annexin A2 (ANXA2) is a protein implicated in diverse cellular functions, including exocytosis, DNA synthesis and cell proliferation. It was recently proposed to be involved in RNA metabolism because it was shown to associate with some cellular mRNA. Here, we identified ANXA2 as a RNA binding protein (RBP) that binds IBV (Infectious Bronchitis Virus) pseudoknot RNA. We first confirmed the binding of ANXA2 to IBV pseudoknot RNA by ultraviolet crosslinking and showed its binding to RNA pseudoknot with ANXA2 protein in vitro and in the cells. Since the RNA pseudoknot located in the frameshifting region of IBV was used as bait for cellular RBPs, we tested whether ANXA2 could regulate the frameshfting of IBV pseudoknot RNA by dual luciferase assay. Overexpression of ANXA2 significantly reduced the frameshifting efficiency from IBV pseudoknot RNA and knockdown of the protein strikingly increased the frameshifting efficiency. The results suggest that ANXA2 is a cellular RBP that can modulate the frameshifting efficiency of viral RNA, enabling it to act as an anti-viral cellular protein, and hinting at roles in RNA metabolism for other cellular mRNAs

Revenue volatility: the determinants and consequences

In response to the growing concerns over the recurring state fiscal crises, this dissertation aims to shed light on the determinants and consequences of revenue volatility. To this end, the dissertation specifically addresses two questions. First, it examines how the composition of tax bases varies across states and what effects tax base composition has on the cyclical volatility of tax revenues. With particular focus on two major revenue sources relied upon by state governments, general sales tax and individual income tax, this study develops a measure of revenue volatility and investigates the questions using pooled OLS on state panel data over the sample period from 1992 to 2007. Overall, the empirical analysis finds that there exists a wide variation in both sales and individual income tax across states. Regression results indicate that tax base composition significantly affects revenue volatility, with economic structure and demographic-economic characteristics being controlled for. Specifically, tax exemptions for household necessities (food and clothing) and producer goods are found to have statistically significant effects on sales tax volatility. On the other hand, exemptions for Social Security benefits, public pensions, and long-term capital gains, along with deduction for local tax property tax paid, are significantly related to income tax volatility. Second, this dissertation examines how cyclical changes in tax revenues affect state fiscal behavior in terms of the level of spending and taxation, using a panel data set for state governments over the period of 1992 to 2007. Specifically, the study tests fixed effects models that explain own-source expenditure and overall tax rate as a function of revenue gap, the cyclical component of state tax revenue. Regression results reveal that cyclical changes in tax revenues are positively related to changes in own-source expenditures, whereas they are negatively related to changes in tax rates, suggesting the relationship between revenue volatility and fiscal instability. Based on these findings, the dissertation concludes by discussing the dynamics of state fiscal behavior over the business cycle and suggesting spending-smoothing rules as a policy solution to structural budget deficits and fiscal crises.Ph. D.Includes bibliographical referencesIncludes vitaby Sunjoo Kwa

Diagram of frameshifting reporters.

<p>(A) Structure of dual luciferase reporter constructs with Renilla and Firefly luciferase genes in p2Luc vector. IBV genomic sequence (72 bp) is composed of slippery site, stop codon, spacer linker and pseudoknot region. In-frame and no-frame control reporters are also shown. Frameshifting efficiency was calculated as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0024067#s4" target="_blank">Materials and Methods</a>. (B) Predicted results from the four reporters.</p

ANXA2 regulates dual luciferase reporters.

<p>(A) Dual luciferase reporter assay after overexpression of ANXA2 in HEK293T cells. Relative frameshifting efficiency was measured for the vector and FLAG-tagged ANXA2 transfected cells. Expression of ANXA2 was confirmed by Western blot analysis with anti-ANXA2 antibody. Equal loading of proteins were confirmed by anti-β-actin antibody. (B) Luciferase reporter assay after overexpression of ANXA2 in LNCaP cells. Relative frameshifting efficiency was measured for the vector and FLAG-tagged ANXA2 transfected cells. Expression of ANXA2 was confirmed by Western blot analysis with anti-ANXA2 antibody. Anti-β-actin antibody was used as a loading control. (C) Diagram of siRNA or shRNA target sequences (dashed line) in the coding regions ANXA2 mRNA (filled line). Locations of a shRNA and two siRNAs were indicated (shANX2-1: 66–83 nt, siANX2-2: 109–129 nt, siANX2-3: 772-792 nt). (D) Dual luciferase reporter assay following knockdown of ANXA2 in HEK293T cells. Relative frameshifting efficiency was measured with pSUPER vector (Vector) and with pSUPER-ANXA2 (shANX2-1) transfected cells. Western blot analysis was performed after shRNA transfection. Anti-β-actin antibody was used as a loading control. (E) Luciferase reporter assay after knockdown of ANXA2 in HEK293T cells. Relative frameshifting efficiency was measured from control siRNA (siGFP) or two independent ANXA2 siRNAs (siANX2-2 and -3) transfected cells. Western blot analysis was performed after siRNA transfection. Anti-β-actin antibody was used as a loading control. In (A), (B), (D) and (E), the experiments were performed in triplicate and mean ± s.d. are shown.</p

ANXA2 specifically binds to pseudoknot RNA.

<p>(A) GST pull-down analysis of IBV RNA. Wild-type (WT) and mutant (MT) IBV pseudoknot RNA were labeled with [γ-³²P] ATP and mixed with GST or GST-ANXA2 protein. Each sample was pulled down with Glutathione 4B Sepharose and pelleted radioactivity was measured with a scintillation counter. Three independent experiments were performed and statistical analysis was done. All experiments were performed in triplicate and mean ± s.d. are shown. ***: <i>p<0.001</i> (B) EMSA with GST or GST-ANXA2 with radiolabeled IBV RNA. Bound and unbound RNA bands are indicated. Lane 1 contains no protein; Lanes 2 and 3 contain GST protein (5 and 10 µM, respectively); Lanes 4-7 contain GST-ANXA2 protein (0.5, 2, 5 and 10 µM, respectively). (C) RNA immunoprecipitation assay. LNCaP cells were co-transfected with reporters (wild-type or mutant IBV pseudoknot plasmid Wild-type or mutant) and ANXA2 expression plasmids (Flag-vector or Flag-ANXA2). After formaldehyde fixation, immunoprecipitations were performed with FLAG-M2 agarose beads. Bound RNA was extracted from the immune complexes and analyzed by RT-PCR (left panel) and qRT-PCR (right panel). PCR products were resolved by electrophoresis in agarose gel and visualized by staining with ethidium bromide. ANXA2 mRNA and IBV PK RNA were also shown as an expression and input controls. Immunoprecipitation of Flag-ANXA2 was confirmed by immunoblotting (IB) using anti-Flag antibody. In the qRT-PCR data, wild-type PK RNA (WT) in immune complex was normalized with input PK RNA level and presented as relative enrichment in comparison to mutant PK RNA (MT). (D) RNA immunoprecipitation assay in HEK293T cells. Reporter plasmids (wild-type or mutant IBV pseudoknot plasmid) were transfected into HEK293T cells and fixed with formaldehyde. Sonicated lysates were then incubated with the antibodies as indicated (anti-IgG or anti-ANXA2). RNA-protein complexes were precipitated with protein-G beads. Bound RNA was extracted from the immune complexes and analyzed by RT-PCR and qRT-PCR. Immunoprecipitated ANXA2 protein was shown by immunoblotting (IB). Relative enrichment of binding was shown as in (C).</p

IBV frameshfting elements with slippery site, spacer and pseudoknot.

<p>(A) Genomic sequences of IBV frameshifting elements. Mutant refers to the mutation of CCCC to GGGG in the pseudoknot without any change in the slippery site and spacer region. (B) Predicted RNA secondary structure drawn by pknotsRG web program (<a href="http://bibiserv.techfak.uni-bielefeld.de/pknotsrg/submission.html" target="_blank">http://bibiserv.techfak.uni-bielefeld.de/pknotsrg/submission.html</a>) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0024067#pone.0024067-Marszalkowski1" target="_blank">[24]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0024067#pone.0024067-Heppell1" target="_blank">[25]</a>. Wild-type IBV RNA has G-C base pairs in the pseudoknot structure (box), whereas mutant IBV RNA does not form the pseudoknot structure.</p

Antibacterial Activity against Clinical Isolates and In Vivo Efficacy of Coralmycins

Coralmycins, such as coralmycin A and DH-coralmycin A, have novel molecular skeletons and have been reported to exhibit potent antibacterial activity against standard Gram-positive bacterial strains. Here, the in vitro antibacterial activity against an extensive clinical isolate collection, time-kill kinetics, pharmacokinetics (PK), and in vivo efficacy of coralmycins were studied. Coralmycin A showed potent antibacterial activity with an MIC90 of 1 mg/L against 73 clinical methicillin-resistant Staphylococcus aureus and coagulase-negative staphylococci isolates, which was 2&ndash;8 times higher than the corresponding activities of DH-coralmycin A, vancomycin, daptomycin, and linezolid, and against 73 vancomycin-resistant Enterococcus and Streptococcus pneumoniae isolates, which was 4&ndash;16 times higher than the corresponding activities of DH-coralmycin A, daptomycin, and linezolid. Pharmacokinetic analysis after i.v. injection showed that coralmycins have a moderate volume of distribution and moderate-to-high clearance in mice. The coralmycin A and DH-coralmycin A bioavailability values were 61.3% and 11.7%, respectively, after s.c. administration. In a mouse respiratory tract infection model, coralmycin A showed bacteriostatic and bactericidal in vivo efficacies at an s.c. administration of 4 and 100 mg/kg bid, respectively; these efficacies were similar to those of vancomycin at 4 and 20 mg/kg bid, respectively. The present findings indicate that coralmycin A has great potential as a new class of antibiotic for treating infections caused by multidrug-resistant Gram-positive bacteria