Characterisation of RNA binding proteins and their roles in the Drosophila germline

The important role of RNA binding proteins (RBPs) in regulating the fate and functions of RNAs has led to the development of transcript-specific as well as transcriptome-wide techniques allowing an unbiased and comprehensive identification of RBPs. These methods have extended our knowledge of the extent of RBPs in a cell, and studying the roles of these newly identified RBPs in cellular processes has provided us with novel insights into the RNA binding mechanisms, functions and regulation of RNA binding proteins. For my PhD work, I assessed the RNA binding functions of two proteins identified in high-throughput screens. The first protein is the Fragile X Mental Retardation protein (FMR1), identified in a transcript-specific pulldown targeted at the Drosophila maternal mRNA oskar. I show that FMR1 is a bona fide component of the oskar RNA-protein complexes that interacts with the oskar 3’UTR in vivo. FMR1 positively regulates Oskar protein levels in the oocyte, without any effect on oskar RNA levels. Oskar protein nucleates germ plasm assembly and germ cell formation in the embryo, and the reduction in Oskar protein levels leads to a reduction in the number of pole cells formed in embryos knocked down for FMR1. Finally, I tried to determine how FMR1 regulates translation, with roles identified as both a repressor and activator of translation. FMR1 contains two types of RNA binding domains: two KH domains and a C-terminal RGG box. I show that, in vitro, FMR1 activates translation through the KH domains and requires the C-terminal RGG box for repression of translation. I have thus identified a new role of FMR1 in germline development in Drosophila melanogaster, and also a putative mechanism of how FMR1 performs antagonistic functions in translation regulation. The second protein I studied is the microtubule binding protein EB1, identified as a putative RNA binding protein in a transcriptome-wide RNA interactome capture study performed in Drosophila embryos. Preliminary data showed that EB1 binds to polyU25 RNA in vitro, and uses the same binding surface for interacting with microtubules and RNA. I show that EB1 binds to microtubules and RNA in a mutually exclusive manner in vitro. Furthermore, I performed a RIP-seq experiment to identify the in vivo targets of EB1, but failed to validate the interaction of any of the top candidates with EB1 in vivo. This does not, however, negate a role of EB1 as an RNA binding protein altogether, as RNA might be regulating the functions of the protein, and this would require further investigation

Macropinocytosis confers resistance to therapies targeting cancer anabolism.

Macropinocytic cancer cells scavenge amino acids from extracellular proteins. Here, we show that consuming necrotic cell debris via macropinocytosis (necrocytosis) offers additional anabolic benefits. A click chemistry-based flux assay reveals that necrocytosis provides not only amino acids, but sugars, fatty acids and nucleotides for biosynthesis, conferring resistance to therapies targeting anabolic pathways. Indeed, necrotic cell debris allow macropinocytic breast and prostate cancer cells to proliferate, despite fatty acid synthase inhibition. Standard therapies such as gemcitabine, 5-fluorouracil (5-FU), doxorubicin and gamma-irradiation directly or indirectly target nucleotide biosynthesis, creating stress that is relieved by scavenged nucleotides. Strikingly, necrotic debris also render macropinocytic, but not non-macropinocytic, pancreas and breast cancer cells resistant to these treatments. Selective, genetic inhibition of macropinocytosis confirms that necrocytosis both supports tumor growth and limits the effectiveness of 5-FU in vivo. Therefore, this study establishes necrocytosis as a mechanism for drug resistance

Effect of EMS on morpho-physiological characters of wheat in reference to stay green trait

To feed the ever growing world population, the demand of food supply must be increased by 70 % of major cereal crops like wheat, rice etc. It was predicted that the detrimental effect of abiotic stresses like drought, heat, salt etc. on yield would be decreased by genetic improvement in terms of photosynthetic response, long green leaf duration and delayed leaf senescence. ‘Stay green’ is a vital trait of all crops which is directly associated with the capacity of the plant to maintain CO2 assimilation, photosynthesis and chlorophyll content. The present study was conducted to develop the stay green mutants genotype by using 1.5 % Ethyl Methane Sulphonate (EMS). The variety K 7410 showed highest leaf area 37.34 cm2, seeds per spike 65.47, 1000 grain weight 62.03 g after treatment of EMS among morphological characters observed. Among physiological characters of wheat variety Sonalik showed lowest RWC (21.48 %), HD 2135 showed lowest chlorophyll content (33.53 µg/cm2) and C 306 showed lowest photosynthetic rate (15.05 µmol/m2sec) after treatment of EMS. But varieties K 7410, VL 401 and RAJ 3765 varieties showed higher value of physiological characters after the treatment. The results suggested that the stay green trait had been developed by mutation (EMS) in these three wheat varieties and they can exhibit better tolerance under abiotic stress conditions like drought, high temperature. Such results would prove useful for further research and crop management in stress affected areas or under unfavourable climatic conditions