Characterizing the localization and role of lin-5 and era-1 mRNAs in early C. elegans embryos

Asymmetric cell division is essential for the generation of diversity during development and the function of stem cell lineages. The Caenorhabditis elegans zygote is an attractive model to investigate the mechanisms of spindle positioning during asymmetric cell division. In this polarized cell, the asymmetric distribution of cortical force generators along the antero-posterior axis and pulling on astral microtubules leads to the unequal cleavage of the one-cell embryo. The mechanisms underlying such cortical force generation are thought to act strictly at the protein level. In this thesis work we report that the mRNA encoding the cortical force generator component LIN-5 is enriched around centrosomes in early embryos, in a manner that depends on microtubules and dynein. We established that the lin-5 coding sequence is necessary and sufficient for mRNA enrichment around centrosomes in C. elegans. In addition, we found that lin-5 mRNA is mislocalized in lin-5(ev571) mutant embryos, which harbor a 9 nucleotide insertion in the coding sequence. Moreover, an intragenic revertant of lin-5(ev571), lin-5(ev571he63), also exhibits mislocalized lin-5 mRNA distribution. We demonstrated that this is accompanied by diminished pulling forces on the posterior spindle pole, suggesting that centrosomal localization of lin-5 mRNA is important for robust pulling forces. We found also that lin-5 mRNA centrosomal enrichment is slightly asymmetric during anaphase, with more transcripts present on the anterior side. We developed a novel FRAP-based assay, which revealed that lin-5 is translated/folded preferentially in the cytoplasm compared to centrosomes. Furthermore, we found that morpholino-mediated inhibition of lin-5 translation diminishes pulling forces on the posterior side during anaphase. Together, these findings lead us to propose that preferential translation/folding of lin-5 in the posterior cytoplasm following release of the mRNA from the posterior centrosome contributes to asymmetric cortical distribution of force generators, and thus to proper spindle positioning. Moreover, we found that the mRNA of an uncharacterized gene, era-1 is enriched on the anterior side of the zygote and is inherited by the anterior blastomeres. Similar to era-1 mRNA, a YFP fusion of ERA-1 protein is also asymmetrically distributed. Moreover, asymmetric distribution of both era-1 mRNA and YFP-ERA-1 protein requires the era-1 3'UTR. Furthermore, the RNA-binding protein MEX-5 is needed for both asymmetric era-1 mRNA localization and for its translational activation. Furthermore, we report that the clathrin heavy chain CHC-1 negatively regulates pulling forces acting on centrosomes during interphase and on spindle poles during mitosis in one-cell C. elegans embryos. We establish a similar role for the cytokinesis/apoptosis/RNA-binding protein CAR-1 and uncover that CAR-1 is needed to maintain normal levels of CHC-1. We demonstrate that CHC-1 is necessary for proper organization of the cortical acto-myosin network and for full cortical tension. Furthermore, we establish that the centrosome positioning phenotype of embryos depleted of CHC-1 is alleviated by stabilizing the acto-myosin network. Conversely, we demonstrate that slight perturbations of the acto-myosin network results in excess centrosome movements. Overall, our findings lead us to propose that clathrin plays a critical role in centrosome positioning by promoting acto-myosin cortical tension

Uniformly curated signaling pathways reveal tissue-specific cross-talks and support drug target discovery

Motivation: Signaling pathways control a large variety of cellular processes. However, currently, even within the same database signaling pathways are often curated at different levels of detail. This makes comparative and cross-talk analyses difficult. Results: We present SignaLink, a database containing 8 major signaling pathways from Caenorhabditis elegans, Drosophila melanogaster, and humans. Based on 170 review and approx. 800 research articles, we have compiled pathways with semi-automatic searches and uniform, well-documented curation rules. We found that in humans any two of the 8 pathways can cross-talk. We quantified the possible tissue- and cancer-specific activity of cross-talks and found pathway-specific expression profiles. In addition, we identified 327 proteins relevant for drug target discovery. Conclusions: We provide a novel resource for comparative and cross-talk analyses of signaling pathways. The identified multi-pathway and tissue-specific cross-talks contribute to the understanding of the signaling complexity in health and disease and underscore its importance in network-based drug target selection. Availability: http://SignaLink.orgComment: 9 pages, 4 figures, 2 tables and a supplementary info with 5 Figures and 13 Table

RNA Interference Links Oxidative Stress to the Inhibition of Heat Stress Adaptation

Increased oxidative stress is associated with various diseases and aging, while adaptation to heat stress is an important determinant of survival and contributes to longevity. However, the impact of oxidative stress on heat resistance remains largely unclear. Aim: In this study we investigated how oxidative stress impinges on heat stress responses. Results: We report that hydrogen-peroxide (H2O2) pretreatment inhibits both acquired thermotolerance and heat-induced Hsp70 expression in mammalian cells, as well as acquired thermotolerance in the nematode Caenorhabditis elegans, via RNA interference. Moreover, we demonstrate that elimination of RNA interference by silencing key enzymes in microRNA biogenesis, dcr-1 or pash-1, restores the diminished intrinsic thermotolerance of aged and H 2O 2-elimination compromised (catalase-2 and peroxiredoxin-2 deficient) worms. Innovation and Conclusion: These results uncover a novel post-transcriptional element in the regulation of heat stress adaptation under oxidative conditions that may have implications in disease susceptibility and aging. Antioxid. Redox Signal. 17, 890–901