Strong Eukaryotic IRESs Have Weak Secondary Structure

BACKGROUND: The objective of this work was to investigate the hypothesis that eukaryotic Internal Ribosome Entry Sites (IRES) lack secondary structure and to examine the generality of the hypothesis. METHODOLOGY/PRINCIPAL FINDINGS: IRESs of the yeast and the fruit fly are located in the 5'UTR immediately upstream of the initiation codon. The minimum folding energy (MFE) of 60 nt RNA segments immediately upstream of the initiation codons was calculated as a proxy of secondary structure stability. MFE of the reverse complements of these 60 nt segments was also calculated. The relationship between MFE and empirically determined IRES activity was investigated to test the hypothesis that strong IRES activity is associated with weak secondary structure. We show that IRES activity in the yeast and the fruit fly correlates strongly with the structural stability, with highest IRES activity found in RNA segments that exhibit the weakest secondary structure. CONCLUSIONS: We found that a subset of eukaryotic IRESs exhibits very low secondary structure in the 5'-UTR sequences immediately upstream of the initiation codon. The consistency in results between the yeast and the fruit fly suggests a possible shared mechanism of cap-independent translation initiation that relies on an unstructured RNA segment

Thiram inhibits angiogenesis and slows the development of experimental tumours in mice

Thiram-tetramethylthiuram disulphide – a chelator of heavy metals, inhibited DNA synthesis and induced apoptosis in cultured bovine capillary endothelial cells. Bovine capillary endothelial cells were 10–60-fold more sensitive to thiram than other cell types. These effects were prevented by addition of antioxidants, indicating involvement of reactive oxygen species. Exogenously added Cu2+ impeded specifically and almost completely the inhibitory effect of thiram for bovine capillary endothelial cells. Moreover, thiram had markedly inhibited human recombinant Cu/Zn superoxide dismutase enzymatic activity (85%) in vitro. Moreover, PC12-SOD cells with elevated Cu/Zn superoxide dismutase were less sensitive to thiram treatment than control cells. These data indicate that the effects of thiram are mediated by inhibition of Cu/Zn superoxide dismutase activity. Oral administration of thiram (13–30 μg mouse−1), inhibited angiogenesis in CD1 nude mice. Tumour development is known to largely depend on angiogenesis. We found that oral administration of thiram (30 μg) to mice caused significant inhibition of C6 glioma tumour development (60%) and marked reduction (by 3–5-fold) in metastatic growth of Lewis lung carcinoma. The data establish thiram as a potential inhibitor of angiogenesis and raise the possibility for its use as therapy in pathologies in which neovascularisation is involved, including neoplasia

An Internal Ribosome Entry Site Directs Translation of the 39-Gene from Pelargonium Flower Break Virus Genomic RNA: Implications for Infectivity

[EN] Pelargonium flower break virus (PFBV, genus Carmovirus) has a single-stranded positive-sense genomic RNA (gRNA) which contains five ORFs. The two 59-proximal ORFs encode the replicases, two internal ORFs encode movement proteins, and the 39-proximal ORF encodes a polypeptide (p37) which plays a dual role as capsid protein and as suppressor of RNA silencing. Like other members of family Tombusviridae, carmoviruses express ORFs that are not 59-proximal from subgenomic RNAs. However, in one case, corresponding to Hisbiscus chlorotic ringspot virus, it has been reported that the 39-proximal gene can be translated from the gRNA through an internal ribosome entry site (IRES). Here we show that PFBV also holds an IRES that mediates production of p37 from the gRNA, raising the question of whether this translation strategy may be conserved in the genus. The PFBV IRES was functional both in vitro and in vivo and either in the viral context or when inserted into synthetic bicistronic constructs. Through deletion and mutagenesis studies we have found that the IRES is contained within a 80 nt segment and have identified some structural traits that influence IRES function. Interestingly, mutations that diminish IRES activity strongly reduced the infectivity of the virus while the progress of the infection was favoured by mutations potentiating such activity. These results support the biological significance of the IRES-driven p37 translation and suggest that production of the silencing suppressor from the gRNA might allow the virus to early counteract the defence response of the host, thus facilitating pathogen multiplication and spread.This research was supported by grants BFU2006-11230 and BFU2009-11699 from the Spanish Ministerio de Ciencia e Innovacion (MICINN) and by grants ACOM/2006/210 and ACOMP/2009/040 (to CH) and GVPRE/2008/121 (to OF-M) from the Generalitat Valenciana. The latter was the recipient of an I3P postdoctoral contract from the Spanish Consejo Superior de Investigaciones Cientificas and an additional contract from MICINN. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.Fernandez Miragall, O.; Hernandez Fort, C. (2011). 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