Degradation of YRA1 Pre-mRNA in the Cytoplasm Requires Translational Repression, Multiple Modular Intronic Elements, Edc3p, and Mex67p

The yeast YRA1 pre-mRNA contains multiple intronic elements that regulate transcript decay and translatability via the Edc3p decapping activator and the Mex67p/Mtr2p export receptor

microRNA-Mediated Messenger RNA Deadenylation Contributes to Translational Repression in Mammalian Cells

Animal microRNAs (miRNAs) typically regulate gene expression by binding to partially complementary target sites in the 3′ untranslated region (UTR) of messenger RNA (mRNA) reducing its translation and stability. They also commonly induce shortening of the mRNA 3′ poly(A) tail, which contributes to their mRNA decay promoting function. The relationship between miRNA-mediated deadenylation and translational repression has been less clear. Using transfection of reporter constructs carrying three imperfectly matching let-7 target sites in the 3′ UTR into mammalian cells we observe rapid target mRNA deadenylation that precedes measureable translational repression by endogenous let-7 miRNA. Depleting cells of the argonaute co-factors RCK or TNRC6A can impair let-7-mediated repression despite ongoing mRNA deadenylation, indicating that deadenylation alone is not sufficient to effect full repression. Nevertheless, the magnitude of translational repression by let-7 is diminished when the target reporter lacks a poly(A) tail. Employing an antisense strategy to block deadenylation of target mRNA with poly(A) tail also partially impairs translational repression. On the one hand, these experiments confirm that tail removal by deadenylation is not strictly required for translational repression. On the other hand they show directly that deadenylation can augment miRNA-mediated translational repression in mammalian cells beyond stimulating mRNA decay. Taken together with published work, these results suggest a dual role of deadenylation in miRNA function: it contributes to translational repression as well as mRNA decay and is thus critically involved in establishing the quantitatively appropriate physiological response to miRNAs

Global Mapping of DNA Conformational Flexibility on Saccharomyces cerevisiae

In this study we provide the first comprehensive map of DNA conformational flexibility in Saccharomyces cerevisiae complete genome. Flexibility plays a key role in DNA supercoiling and DNA/protein binding, regulating DNA transcription, replication or repair. Specific interest in flexibility analysis concerns its relationship with human genome instability. Enrichment in flexible sequences has been detected in unstable regions of human genome defined fragile sites, where genes map and carry frequent deletions and rearrangements in cancer. Flexible sequences have been suggested to be the determinants of fragile gene proneness to breakage; however, their actual role and properties remain elusive. Our in silico analysis carried out genome-wide via the StabFlex algorithm, shows the conserved presence of highly flexible regions in budding yeast genome as well as in genomes of other Saccharomyces sensu stricto species. Flexibile peaks in S. cerevisiae identify 175 ORFs mapping on their 3’UTR, a region affecting mRNA translation, localization and stability. (TA)n repeats of different extension shape the central structure of peaks and co-localize with polyadenylation efficiency element (EE) signals. ORFs with flexible peaks share common features. Transcripts are characterized by decreased half-life: this is considered peculiar of genes involved in regulatory systems with high turnover; consistently, their function affects biological processes such as cell cycle regulation or stress response. Our findings support the functional importance of flexibility peaks, suggesting that the flexible sequence may be derived by an expansion of canonical TAYRTA polyadenylation efficiency element. The flexible (TA)n repeat amplification could be the outcome of an evolutionary neofunctionalization leading to a differential 3’-end processing and expression regulation in genes with peculiar function. Our study provides a new support to the functional role of flexibility in genomes and a strategy for its characterization inside human fragile sites

A breakthrough on Amanita phalloides poisoning: an effective antidotal effect by polymyxin B

Amanita phalloides is responsible for more than 90 % of mushroom-related fatalities, and no effective antidote is available. a-Amanitin, the main toxin of A. phalloides, inhibits RNA polymerase II (RNAP II), causing hepatic and kidney failure. In silico studies included docking and molecular dynamics simulation coupled to molecular mechanics with generalized Born and surface area method energy decomposition on RNAP II. They were performed with a clinical drug that shares chemical similarities to a-amanitin, polymyxin B. The results show that polymyxin B potentially binds to RNAP II in the same interface of a-amanitin, preventing the toxin from binding to RNAP II. In vivo, the inhibition of the mRNA transcripts elicited by a-amanitin was efficiently reverted by polymyxin B in the kidneys. Moreover, polymyxin B significantly decreased the hepatic and renal a-amanitin-induced injury as seen by the histology and hepatic aminotransferases plasma data. In the survival assay, all animals exposed to a-amanitin died within 5 days, whereas 50 % survived up to 30 days when polymyxin B was administered 4, 8, and 12 h post-a-amanitin. Moreover, a single dose of polymyxin B administered concomitantly with a-amanitin was able to guarantee 100 % survival. Polymyxin B protects RNAP II from inactivation leading to an effective prevention of organ damage and increasing survival in a-amanitin-treated animals. The present use of clinically relevant concentrations of an already human-use-approved drug prompts the use of polymyxin B as an antidote for A. phalloides poisoning in humans.Juliana Garcia, Vera Marisa Costa, Ricardo Dinis-Oliveira and Ricardo Silvestre thank FCT-Foundation for Science and Technology-for their PhD grant (SFRH/BD/74979/2010), Post-doc grants (SFRH/BPD/63746/2009 and SFRH/BPD/110001/2015) and Investigator grants (IF/01147/2013) and (IF/00021/2014), respectively. This work was supported by the Fundacao para a Ciencia e Tecnologia (FCT) - project PTDC/DTPFTO/4973/2014 - and the European Union (FEDER funds through COMPETE) and National Funds (FCT, Fundacao para a Ciencia e Tecnologia) through project Pest-C/EQB/LA0006/2013

Evolutionary history of the OmpR/IIIA family of signal transduction two component systems in Lactobacillaceae and Leuconostocaceae

<p>Abstract</p> <p>Background</p> <p>Two component systems (TCS) are signal transduction pathways which typically consist of a sensor histidine kinase (HK) and a response regulator (RR). In this study, we have analyzed the evolution of TCS of the OmpR/IIIA family in <it>Lactobacillaceae </it>and <it>Leuconostocaceae</it>, two families belonging to the group of lactic acid bacteria (LAB). LAB colonize nutrient-rich environments such as foodstuffs, plant materials and the gastrointestinal tract of animals thus driving the study of this group of both basic and applied interest.</p> <p>Results</p> <p>The genomes of 19 strains belonging to 16 different species have been analyzed. The number of TCS encoded by the strains considered in this study varied between 4 in <it>Lactobacillus helveticus </it>and 17 in <it>Lactobacillus casei</it>. The OmpR/IIIA family was the most prevalent in <it>Lactobacillaceae </it>accounting for 71% of the TCS present in this group. The phylogenetic analysis shows that no new TCS of this family has recently evolved in these <it>Lactobacillaceae </it>by either lineage-specific gene expansion or domain shuffling. Furthermore, no clear evidence of non-orthologous replacements of either RR or HK partners has been obtained, thus indicating that coevolution of cognate RR and HKs has been prevalent in <it>Lactobacillaceae</it>.</p> <p>Conclusions</p> <p>The results obtained suggest that vertical inheritance of TCS present in the last common ancestor and lineage-specific gene losses appear as the main evolutionary forces involved in their evolution in <it>Lactobacillaceae</it>, although some HGT events cannot be ruled out. This would agree with the genomic analyses of <it>Lactobacillales </it>which show that gene losses have been a major trend in the evolution of this group.</p

School Effects on the Wellbeing of Children and Adolescents

Well-being is a multidimensional construct, with psychological, physical and social components. As theoretical basis to help understand this concept and how it relates to school, we propose the Self-Determination Theory, which contends that self-determined motivation and personality integration, growth and well-being are dependent on a healthy balance of three innate psychological needs of autonomy, relatedness and competence. Thus, current indicators involve school effects on children’s well-being, in many diverse modalities which have been explored. Some are described in this chapter, mainly: the importance of peer relationships; the benefits of friendship; the effects of schools in conjunction with some forms of family influence; the school climate in terms of safety and physical ecology; the relevance of the teacher input; the school goal structure and the implementation of cooperative learning. All these parameters have an influence in promoting optimal functioning among children and increasing their well-being by meeting the above mentioned needs. The empirical support for the importance of schools indicates significant small effects, which often translate into important real-life effects as it is admitted at present. The conclusion is that schools do make a difference in children’s peer relationships and well-being