Functional analysis of Pumilio proteins in vitro and in vivo

In neurons, a subset of mRNAs localizes as large ribonucleoprotein particles (RNPs) to the dendritic compartment. The subsequent activation of translation upon specific inputs, such as synaptic activity or neurotrophins, allows for the modulation of the strength of existing synapses via changes in their electrical properties and morphological rearrangement of dendritic spines. These processes are thought to form the basis of such fundamental activities as learning and memory. One key feature of mRNA localization is that localization precedes translation. As a consequence, RNA has to be kept translationally silent during its transport towards the proper target compartment. Whether this two-step process, RNA transport and translational regulation, shares common protein components is currently unclear. Potential candidates for such a dual role are the Pumilio (Pum) proteins. In the Drosophila oocyte, the Pum gene encodes for a protein that is known to act as an inhibitor of translation. In mammals, two Pum genes, Pum1 and Pum2, exist. However, the role of these proteins in mammals and particularly in neurons has not been unravelled. We could recently show that Pum2 is present in RNPs in the cell body and in dendrites of hippocampal neurons, suggesting that mammalian Pum proteins might be part of the dendritic RNA transport machinery in neurons. Both protein and RNA components of neuronal Pum-containing RNPs have not been characterized yet. The ultimate goal of this proposal is to investigate whether Pum proteins exert a dual role in RNA transport and/or tranlsational control in neurons. Using a biochemical approach, we will characterize both the protein and mRNA components of Pum-containing RNPs in rat brain. We will then determine whether Pum downregulation affects dendritic RNA transport. Since Pum is involved in translational repression of several mRNAs in different organisms, we will investigate whether Pum may act on dendritically localized mRNAs using in vitro and in vivo reporter assays. Finally, to investigate the role of Pum proteins in the brain, we will create tissue-specific knockdowns in rats for Pum1+2 proteins. Conditional RNAi will allow inducible and cell type-specific gene silencing in rats to dissect the gene function in vivo. The results of these studies will contribute to the understanding and highlight common molecular interactions between the dendritic RNA transport and translational control machineries in neurons.

Learning and the subsequent storage of memories is thought to occur at the contact sites - the synapses - between nerve cells. However, the molecular mechanisms how this takes place, are largely unknown. One attractive and largely accepted hypothesis is the localization of a subset of messenger RNAs to synapses. Once a particular synapse has been activated repetitively - a requisite for associative memory - then the localized transcripts become translated locally into proteins which in turn leads to a modulation of the strength of existing synapses via changes in their electrical properties and their morphological rearrangement. Potential candidates for such a dual role in both mRNA localization as well as translational control are the Pumilio (Pum) proteins. In the Drosophila oocyte, the Pum gene encodes for a protein that is known to act as an inhibitor of translation and that is crucial for odor avoidance, an established form of memory in the fly. In mammals, two Pum genes, Pum1 and Pum2, exist. During this project, Paolo Macchi and his team could show that Pum2 protein is present in neuronal RNA granules in the cell body and in dendrites of hippocampal neurons, suggesting that mammalian Pum proteins might be part of the dendritic RNA transport machinery in neurons. Subsequently, we established Pum2 as translational repressor at the synapse demonstrating that Pum2 is not only important for translational control of transcripts, e.g. eIF4E and others, but also for synapse morphogenesis and function in mature neurons.