"You have made your way from worm to man, and much within you is still a worm" (Nieztsche)
Uncovering a new mechanism of cep-1/p53 regulation
After accepting my position in Dundee, we uncovered a novel mechanism of p53 regulation by posttranslational repression and finished those studies in Dundee (link to paper). As part of our focus on p53/cep-1, using genetic screening procedures, we identified a mutant (op236) which leads to hyper activation of worm cep-1/p53 signalling. We found that the evolutionarily conserved gld-1 gene is affected by the op236 mutation and that in the gld-1(op236) mutant, cep-1/p53 dependent transcription of pro-apoptotic genes is up-regulated. gld-1 acts genetically at or above the level of cep-1/p53 and gld-1 directly binds to the cep-1/p53 3'UTR. Mutant gld-1(op236) protein has reduced binding to cep-1/p53 mRNA whereas it binds to other gld-1 targets normally. In gld-1 loss-of-function mutants and upon replacing the cep-1 3'UTR with the unrelated let-858 3'UTR, cep-1/p53 protein levels are dramatically increased. We thus demonstrated a novel mechanism of p53 regulation through gld-1 mediated translational repression. Currently, we are engaged in two follow up projects (see below). My Post Doc Rachael screened for further mutants negatively regulating cep-1/p53 and implicated MAP kinase signalling to affect cep-1/p53 dependent apoptosis. In addition we have several further mutants leading to excessive IR induced germ cell apoptosis and will try to positionally clone and characterize them in the next years.
Combined Genetic and Biochemical approach to understand differential substrate recognition and the model of action of the gld-1 translational repressor.
We are in the first year of a Wellcome Trust funded project aiming to understood how gld-1 can differentially repress different target mRNAs and how gld-1, which we found to translationally repress cep-1/p53 functions as a translational inhibitor (link to paper). gld-1 is part of a conserved family of the signal transduction and activation of RNA (STAR) KH domain containing mRNA binding proteins, which are encoded by C. elegans gld-1 and mammalian quaking genes. quaking is expressed in multiple isoforms in vertebrates and is required for early embryogenesis, myelination and oligodendrocyte differentiation. gld-1 is a cytoplasmic protein mostly located in the centre of the C. elegans gonad. It acts as a translational repressor of multiple target mRNAs. While gld-1 null alleles are defective in the differentiation of mitotic germ into meiotic germ cells, separation of function alleles of gld-1 are defective in distinct aspects of germ cell development, such as the differentiation into male or female germ cells and the proper differentiation of meiotic pachytene cells. Importantly, our studies that link cep-1/p53 regulation to gld-1, as well as previous studies, described gld-1 alleles where separation of function phenotypes were correlated with gld-1 mutants losing binding to a single target mRNA, whereas other tested gld-1 mRNA targets still bound normally.
My PhD student Alper started to use the weak gld-1 (op236) temperature sensitive allele for genetic suppressor and enhancer screens to ultimately identify cofactors of gld-1 and to find direct effectors of gld-1 mediated translational repression. We have isolated and backcrossed several intragenic and extragenic suppressors of the sterile phenotype of gld-1 (op236) worms kept at 25C, and we are currently starting to map the strongest extragenic suppressors. In addition, Alper performed a genome wide RNAi feeding screen for genes that upon inactivation by RNAi lead to synthetic lethality in the gld-1 (op236) background at the permissive temperature of 20C, while not being lethal when inactivated in a wild type background. Alper is currently validating ~50 candidates and we will order knockout mutations corresponding to the most promising candidate genes shortly. In parallel to the genetic approach my Post Doc Ashley Craig started to generate reagents to biochemically purify gld-1 and associated proteins. We hope that proteins we find in our biochemical approach will also turn up in our genetic screens.
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