Dokument

Summary:
The proper development, differentiation and plasticity of the nervous system require an accurate regulation at multiple levels of gene expression. One important class of post-transcriptional regulators are microRNAs (miRNAs), tiny RNA molecules that inhibit protein synthesis of target mRNAs at the level of mRNA translation and stability. MiRNAs have well documented roles in the control of neuronal development and function. Specific miRNAs, such as miR-134, regulate the local dynamic translation of mRNAs at the synapse thereby controlling activity-dependent changes in synaptic strength. MiRNAs regulate mRNA translation within a large RNA-protein complex, the microRNA-induced silencing complex (miRISC). Whereas the core components of miRISC, e.g. Argonaute (Ago) and GW182 proteins, are highly conserved, cell-type specific auxiliary proteins play important roles in the modulation of miRISC activity. The molecular mechanisms by which miRISC activity is specifically regulated in the neuronal system by such auxiliary proteins however are poorly described. This project presents the validation and evaluation of the first large scale screening study that was performed in order to find novel RNA-binding proteins (RBPs) that regulate miRISC activity in primary neurons. The RNAi-based screen identified the RBPs Nova1, Ncoa3 and Ewsr1 as new modulators of miRISC in neurons and further confirmed the function of two previously reported miRISC interacting proteins (Ddx6, Tnrc6c). Subsequently, Nova1 was chosen for follow-up experiments directed at the elucidation of the underlying molecular mechanisms. Using luciferase reporter assays for targets of multiple neuronal miRNAs in addition to miR-134, I obtained evidence that Nova1 regulates miRNA activity in neurons in a general manner. In addition, I found that Nova1 can act as a regulator of miRNA activity irrespective of the 3‘UTR context. Further data demonstrated that Nova1 interacts with Ago and the miR-134 target mRNA Limk1. Investigations on the functional relevance of this mechanism revealed that Nova1 is required for miR-134 mediated spine size reduction. Furthermore, I could show that Nova1 is necessary for the upregulation of Limk1 translation upon treatment with brain-derived neurotrophic factor (BDNF), suggesting that Nova1 could be involved in the stimulus-dependent control of neuronal mRNAs. In summary, Nova1 was identified as a new modulator of miRISC activity in neurons and the underlying mechanism was characterized in detail. The data obtained during the thesis project suggests that Nova1 is involved in the dynamic activity-dependent regulation of miRNA function in neurons.

Bibliographie / References

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