Altered expression of plant lysyl tRNA synthetase promotes tRNA misacylation and translational recoding of lysine

The Arabidopsis thaliana lysyl tRNA synthetase (AtKRS) structurally and functionally resembles the well-characterized prokaryotic class IIb KRS, including the propensity to aminoacylate tRNA(Lys) with suboptimal identity elements, as well as non-cognate tRNAs. Transient expression of AtKRS in carrot cells promotes aminoacylation of such tRNAs in vivo and translational recoding of lysine at nonsense codons. Stable expression of AtKRS in Zea mays causes translational recoding of lysine into zeins, significantly enriching the lysine content of grain.     

The protein synthesis inhibitors,oxazolidinones and chloramphenicol,cause extensive translational inaccuracy in vivo

The oxazolidinone family is a new class of synthetic antibiotics that bind to the bacterial 50S ribosomal subunit. Two members of the family, linezolid and XA043, were examined for their effects on translational fidelity using a lacZ reporter gene in vivo. Both promoted highly significant frameshifting and nonsense suppression. Chloramphenicol, a peptidyl transferase inhibitor, affected translational fidelity in a similar fashion. Neither the oxazolidinones nor chloramphenicol stimulated misincorporation of amino acid residues at position 461 in the lacZ gene. In contrast, the aminoglycosides gentamicin and paromomycin, which interact with the decoding region of the 30S subunit, caused significant misincorporation but only modest increases in frameshifting or stop codon readthrough of the lacZ gene. We conclude that effects on translational fidelity may play a significant role in the mechanism of action of the oxazolidinones.     

Regulated expression of plant tRNA genes by the prokaryotic tet and lac repressors

The prokaryotic tet operator (tetO) sequence was inserted at positions upstream and downstream of sequences encoding the Arabidopsis thaliana tRNA _AUC ^Lys or tRNA _AUC ^Trp suppressor tRNAs, and tRNA expression in carrot protoplasts was measured by translational suppression of a nonsense codon in a luciferase reporter gene. Regulation of tRNA expression by the tetracycline repressor (tetR) occurred from genes with the tetO inserted at position –1 (for the tRNA _AUC ^Trp gene), or at positions –2, –6 and –10 (for the tRNA _AUC ^Lys gene), and repression reached 90%. The inducer tetracycline (Tc) restored tRNA expression. Similarly, carrot protoplasts transfected with human tRNA _AUC ^Ser genes containing the lac operator (lacO) in their 5-flanking sequence with or without the lac repressor (lacI) gene, conditionally expressed tRNAs which suppressed the luciferase reporter. Up to 30-fold repression occured by the lactose repressor when lacO was located at position –1 of the tRNA _AUC ^Ser coding sequence. In the presence of the inducer isopropyl--thiogalactoside (IPTG), repression was relieved. These results demonstrate that sequences flanking tRNA genes can strongly influence tRNA expression in plants, and in a conditional fashion when bound by inducible proteins.     

Ribosomal suppressors and antisuppressors in Podospora anserina: Altered susceptibility to paromomycin and relationships between genetic and phenotypic suppression

Informational suppressors and antisuppressors have been previously isolated in Podospora anserina, and their properties suggest that they could be ribosomal mutants involved in the control of translational fidelity. In this paper we present results concerning relationships between these mutants and paromomycin, an aminoglycoside antibiotic known to stimulate translational errors. The mutants were found to manifest an altered growth sensitivity to this drug as compared with the wild-type strain: Most of the suppressors were more sensitive and, in contrast, most of the antisuppressors were more resistant to paromomycin. Moreover, phenotypic suppression of an auxotrophic mutation by paromomycin was observed only if a suppressor and an antisuppressor had been introduced in the strain. These results suggest that ambiguity levels could be altered in the suppressor and antisuppressor strains. In addition, paromomycin was shown to abolish sporulation, which suggests relationships between mistranslation and a step of cellular differentiation.This work was supported by a DGRST grant and by a NATO grant.     

Suppression of Nonsense Mutations in the Dystrophin Gene by a Suppressor tRNA Gene

Nonsense mutations in the dystrophin gene are the cause of Duchenne muscular dystrophy (DMD) in 10–15% of patients. In such an event, one approach to gene therapy for DMD is the use of suppressor tRNAs to overcome the premature termination of translation of the mutant mRNA. We have carried out cotransfection of the HeLa cell culture with constructs containing a suptRNA gene (pcDNA3suptRNA) and a marker LacZ gene (pNTLacZhis) using their polymer VSST-525 complexes. It was found that the number of cells producing -galactosidase depends inversely on the dose of the suptRNA gene. A single in vivo injection of the construct providing for expression of the suptRNAochre gene into mdx mouse muscle resulted in the production of dystrophin in 2.5% of fibers. This suggests that suppressor tRNAs are applicable in gene therapy for hereditary diseases caused by nonsense mutations.     

Contribution of myeloid-derived suppressor cells to tumor-induced immune suppression, angiogenesis, invasion and metastasis

Growing evidence suggests that myeloid-derived suppressor cells (MDSCs), which have been named "immature myeloid cells" or "myeloid suppressor cells" (MSCs), play a critical role during the progression of cancer in tumor-bearing mice and cancer patients. As their name implies, these cells are derived from bone marrow and have a tremendous potential to suppress immune responses. Recent studies indicated that these cells also have a crucial role in tumor progression. MDSCs can directly incorporate into tumor endothelium.They secret many pro-angiogenic factors as well. In addition, they play an essential role in cancer invasion and metastasis through inducing the production of matrix metalloproteinases (MMPs), chemoattractants and creating a pre-metastatic environment. Increasing evidence supports the idea that cancer stem cells (CSCs) are responsible for tumorigenesis, resistance to therapies, invasion and metastasis.Here, we hypothesize that CSCs may "hijack" MDSCs for use as alternative niche cells, leading to the maintenance of stemness and enhanced chemo- and radio-therapy resistance. The countermeasure that directly targets to MDSCs may be useful for against angiogenesis and preventing cancer from invasion and metastasis. Therefore, the study of MDSCs is important to understand tumor progression and to enhance the therapeutic efficacy against cancer.     

Visualizing bz1 missense suppression in Zea mays: an assay for monocot tRNA expression and utilization

Bombardment of a highly expressed dicot tRNA^ala(GAC) gene into Zea mays bz-E2 or bz-E5 coleoptiles causes suppression of an Ala₄₅₈ →Val missense mutation, visualized by the development of anthocyanin pigment. Missense suppression is blocked by mutation of tRNA^ala(GAC) at a site that prevents aminoacylation by the dicot alanyl-tRNA synthetase, indicating that features identified for expression and utilization of dicot tRNAs also function in monocots. This assay of the expression and utilization of tRNA^ala(GAC) also can be used to study a variety of tRNAs and their genes, most of which can be relatively easily altered to be charged by alanyl tRNA synthetase.     

Tolerance and immunity in a mathematical model of T-cell mediated suppression

Regulatory CD4+CD25+ T cells play a major role in natural tolerance to body components and therefore are relevant to understand the self-non-self discrimination by the immune system. The most pressing theoretical question, regarding the fact that these regulatory cells perform their function through linked recognition of the APCs, is how this "non-specific" mechanism permits a proper balance between tolerance and immunity that is compatible with an effective self-non-self discrimination. To tackle this issue, we develop a numerical simulation, which extends a previous mathematical model of T-cell-mediated suppression to include the thymic generation and the peripheral dynamics of many T cell clones. This simulation can mimic the capacity of the immune system to establish natural tolerance to self-antigens and reliably mount immune responses to foreign antigens. Natural tolerance is based on ubiquitous and constitutive self-antigens, which select and sustain clones of specific regulatory cells. Immune responses to foreign antigens are only achieved if they displace the self-antigens from the APCs, leading to a loss of the regulatory cells, and/or if the foreign antigen introduction entails a sharp increase in the total number of APCs. Meaningful behavior is obtained even if differentiation of regulatory cells in the thymus is antigen non-specific, but requires that a minimum number of new T cells enter the periphery per unit of time, and that the repertoire is selected so that anti-self-affinities are within a proper interval. We conclude that positive selection is required to generate a sufficiently high frequency of self-antigen specific regulatory cells that reliably mediate natural tolerance. Negative selection is required to avoid the emergence at the periphery of very high affinity anti-self-regulatory cells that will make the tolerant state so robust that it could no be broken by the introduction of a foreign antigen. This result highlights the importance of repertoire selection in dominant tolerance proposing a novel role for the processes of positive and negative selection within this framework.     

Protein mislocalization in plant cells using a GFP‐binding chromobody

A key challenge in cell biology is to directly link protein localization to function. The green fluorescent protein (GFP)‐binding protein, GBP, is a 13‐kDa soluble protein derived from a llama heavy chain antibody that binds with high affinity to GFP as well as to some GFP variants such as yellow fluorescent protein (YFP). A GBP fusion to the red fluorescent protein (RFP), a molecule termed a chromobody, was previously used to trace in vivo the localization of various animal antigens. In this study, we extend the use of chromobody technology to plant cells and develop several applications for the in vivo study of GFP‐tagged plant proteins. We took advantage of Agrobacterium tumefaciens‐mediated transient expression assays (agroinfiltration) and virus expression vectors (agroinfection) to express functional GBP:RFP fusion (chromobody) in the model plant Nicotiana benthamiana. We showed that the chromobody is effective in binding GFP‐ and YFP‐tagged proteins in planta. Most interestingly, GBP:RFP can be applied to interfere with the function of GFP fusion protein and to mislocalize (trap) GFP fusions to the plant cytoplasm in order to alter the phenotype mediated by the targeted proteins. Chromobody technology, therefore, represents a new alternative technique for protein interference that can directly link localization of plant proteins to in vivo function.     

An NB-LRR protein required for HR signalling mediated by both extra- and intracellular resistance proteins

Tomato (Solanum lycopersicum) Cf resistance genes confer hypersensitive response (HR)-associated resistance to strains of the pathogenic fungus Cladosporium fulvum that express the matching avirulence (Avr) gene. Previously, we identified an Avr4-responsive tomato (ART) gene that is required for Cf-4/Avr4-induced HR in Nicotiana benthamiana as demonstrated by virus-induced gene silencing (VIGS). The gene encodes a CC-NB-LRR type resistance (R) protein analogue that we have designated NRC1 (NB-LRR protein required for HR-associated cell death 1). Here we describe that knock-down of NRC1 in tomato not only affects the Cf-4/Avr4-induced HR but also compromises Cf-4-mediated resistance to C. fulvum. In addition, VIGS using NRC1 in N. benthamiana revealed that this protein is also required for the HR induced by the R proteins Cf-9, LeEix, Pto, Rx and Mi. Transient expression of NRC1(D481V), which encodes a constitutively active NRC1 mutant protein, triggers an elicitor-independent HR. Subsequently, we transiently expressed this auto-activating protein in N. benthamiana silenced for genes known to be involved in HR signalling, thereby allowing NRC1 to be positioned in an HR signalling pathway. We found that NRC1 requires RAR1 and SGT1 to be functional, whereas it does not require NDR1 and EDS1. As the Cf-4 protein requires EDS1 for its function, we hypothesize that NRC1 functions downstream of EDS1. We also found that NRC1 acts upstream of a MAP kinase pathway. We conclude that Cf-mediated resistance signalling requires a downstream NB-LRR protein that also functions in cell death signalling pathways triggered by other R proteins.     

High-yield rapid production of hepatitis B surface antigen in plant leaf by a viral expression system

Recombinant hepatitis B surface antigen (HBsAg) constitutes currently used vaccines against hepatitis B virus, and has been successfully employed as a carrier for foreign epitopes. With the aim of developing an inexpensive, easily administered vaccine source for global immunization, several groups have expressed HBsAg in plant systems. Transgenic plant-derived HBsAg assembles into virus-like particles (VLPs) and is immunogenic in both mice and humans. However, HBsAg expression is relatively low in transgenic plant systems. The time-consuming and labour-intensive process of generating transgenic plants also significantly limits high-throughput analyses of various HBsAg fusion antigens. In this paper, the high-yield rapid production of HBsAg in plant leaf using a novel viral transient expression system is described. Nicotiana benthamiana leaves infiltrated with the MagnICON viral vectors produced HBsAg at high levels, averaging 295 µg/g leaf fresh weight at 10 days post-infection, as measured by a polyclonal enzyme-linked immunosorbent assay. Transiently expressed HBsAg accumulated as the full-length product, formed disulphide-linked dimers, displayed the conformational 'a' antigenic determinant and assembled into VLPs. Immunization of mice with partially purified HBsAg elicited HBsAg-specific antibodies. Furthermore, it was found that transient production of HBsAg using vacuum infiltration of whole plants, rather than syringe infiltration of leaves, was readily scalable, and greatly improved the accumulation of correctly folded HBsAg that displays the protective 'a' determinant.     

Effects of G-quadruplex topology on translational inhibition by tRNA fragments in mammalian and plant systems in vitro

The folding of tRNA fragments (tRFs) into G-quadruplex structures and the implications of G-quadruplexes in translational inhibition have been studied mainly in mammalian systems. To increase our knowledge of these phenomena, we determined the influence of human and plant tRFs and model G-quadruplexes on translation in rabbit reticulocyte lysate and wheat germ extract. The efficiency of translational inhibition in the mammalian system was strongly associated with the type of G-quadruplex topology. In the plant system, the ability of a small RNA to adopt the G-quadruplex conformation was not sufficient to repress translation, indicating the importance of other structural determinants.     

Discrimination between defects in elongation fidelity and termination efficiency provides mechanistic insights into translational readthrough

The suppression of stop codons (termed translational readthrough) can be caused by a decreased accuracy of translation elongation or a reduced efficiency of translation termination. In previous studies, the inability to determine the extent to which each of these distinct processes contributes to a readthrough phenotype has limited our ability to evaluate how defects in the translational machinery influence the overall termination process. Here, we describe the combined use of misincorporation and readthrough reporter systems to determine which of these mechanisms contributes to translational readthrough in Saccharomyces cerevisiae. The misincorporation reporter system was generated by introducing a series of near-cognate mutations into functionally important residues in the firefly luciferase gene. These constructs allowed us to monitor the incidence of elongation errors by monitoring the level of firefly luciferase activity from a mutant allele inactivated by a single missense mutation. In this system, an increase in luciferase activity should reflect an increased level of misincorporation of the wild-type amino acid that provides an estimate of the overall fidelity of translation elongation. Surprisingly, we found that growth in the presence of paromomycin stimulated luciferase activity for only a small subset of the mutant proteins examined. This suggests that the ability of this aminoglycoside to induce elongation errors is limited to a subset of near-cognate mismatches. We also found that a similar bias in near-cognate misreading could be induced by the expression of a mutant form of ribosomal protein (r-protein) S9B or by depletion of r-protein L12. We used this misincorporation reporter in conjunction with a readthrough reporter system to show that alterations at different regions of the ribosome influence elongation fidelity and termination efficiency to different extents.     

Growth suppression of Leydig TM3 cells mediated by aryl hydrocarbon receptor

Exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin induces developmental toxicity in reproductive organs. To elucidate the function of AhR, we generated stable transformants of TM3 cells overexpressing wild-type aryl hydrocarbon receptor (AhR) or its mutants which carried mutations in nuclear localization signal or nuclear export signal. In the presence of 3-methylcholanthrene (MC), proliferation of the cells transfected with wild-type AhR was completely suppressed, whereas cells expressing AhR mutants proliferated in a manner equivalent to control TM3 cells, suggesting AhR-dependent growth inhibition. The suppression was associated with up-regulation of cyclin-dependent kinase inhibitor p21^Cip1, which was abolished by pretreatment with actinomycin D. A p38 MAPK specific inhibitor, SB203580, blocked the increase of p21^Cip1 mRNA in response to MC. Treatment with indigo, another AhR ligand, failed to increase of p21^Cip1 mRNA, although up-regulation of mRNA for CYP1A1 was observed. These data suggest AhR in Leydig cells mediates growth inhibition by inducing p21^Cip1.     

Growth suppression of human transformed cells by treatment with bark extracts from a medicinal plant, Terminalia arjuna

Summary We have investigated the effects of acetone and methanol extracts of a medicinal plant, Terminalia arjuna, on the growth of human normal fibroblasts (WI-38), osteosarcoma (U2OS), and glioblastoma (U251) cells in vitro. We found that both extracts at 30 μg and 60 μg/ml concentrations inhibit the growth of transformed cells; the growth of normal cells was least affected. Although the transformed cells appeared to have fragmented nucleus by Hoechst staining, no deoxyribonucleic acid laddering effect was observed. In response to the extract treatment, the tumor suppressor protein, p53, was induced in U2OS but not in U251 and WI-38 cells. A cyclin-dependent kinase inhibitor, p21^WAF1, was induced in transformed cells only. The study suggests that the bark extract of medicinal plant, T. arjuna, has components that can induce growth arrest of transformed cells by p53-dependent and-independent pathways.