An exploration of the molecular mechanism of neuronal homeostasis : a story of para, Pumilio via NRE?

• Main Author: De, Antara
• Published: University of Warwick 2007
• Subjects: 573.8
• Online Access: http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.491470

The aim of this project was to elucidate the role of the Drosophila translational repressor Pumilio (Pum) in regulating neuronal excitability. It is already known that in the posterior region of the Drosophila embryo Pum acts as a translational repressor of the maternally transcribed hunchback (Izb) gene by interacting with it's 3'Nanos response element (NRE), playing a role in abdominal segmentation. Studies from our lab have shown that the levels of Pum have a positive correlation with synaptic activity but a reciprocal correlation with neuronal excitability. A bioinformatics search has lead to the identification of the similar NRE sequences in the Drosophila sodium channel gene paralytic. (para). I am testing hypothesis that neuronal excitability is dependent on the regulatory activity ofPum; Pum can regulate novel NRE linked messages in an activity-dependent manner I have created transgenic flies expressing lllmclzback NRE-Iuciferase reporter constructs that can respond to different levels of Pum! synaptic activity. I have used these flies to test the hypothesis that changing levels of Pum! activity mediate neuronal homeostasis. This provides strong evidence that Pum! activity can regulate NRE linked mRNAs in the nervous system, but has no direct implication on para. I have mapped the untranslated region (UTR's) of the para transcript(s). I have done this work using RACE (Rapid Amplication of eDNA Ends) analysis and extended PCRs As UTR regions have documented role in localization of transcripts, I have cloned this novel para RACE clones in mGFP vector to study their localization in the nervous system. I have also expressed these clones in over-expression of pumilio background to monitor whether NRE-linked messages can be regulated by pumilio. This will provide in vivo molecular evidence for the action ofPum on para translation. These novel RACE sequences have been cloned into UAS-luciferase vector and transfected into Drosophila Schneider cell line (S2) cells to study their different level of expression.