Investigating the mechanism of Staufen-mediated RNA-localization in mammalian neural stem cells

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Stem cells have the unique ability to divide and produce two daughter cells with different identities. Typically this involves the production of one recurring stem cell and one cell destined for differentiation. In the mammalian brain, this process allows neural stem cells to produce both the neurons and glia of the developing cortex while at the same time ensuring that their own population is replenished. The mechanisms that facilitate these asymmetric cell divisions are poorly understood. Clues can be found in other model organisms such as Drosophila. In fly germ and neural stem cells, it's been demonstrated that certain mRNAs are transported to one half of the dividing cell whereby only one daughter inherits them. Typically, these mRNAs produce proteins that drive the inheriting daughter down the differentiation pathway. Disruption of this mechanism leads to dramatic outcomes in the developing fly, typically rendering they embryo unable to form a proper body plan. During my postdoctoral studies, I asked if this mechanism of asymmetric mRNA localization plays a significant role in the development of the mammalian brain. I demonstrated that the mechanism is, at least in part, conserved in neural stem cells. I found that mRNAs important for conferring a neural lineage, and the proteins involved in their localization, are asymmetrically distributed during differentiating cell divisions. When I disrupted this localizing complex, the cells were no longer able to divide asymmetrically and instead, produced too many neurons at the expense of the stem cell pool. However, two fundamental questions remain. First, at what point are mRNAs destined for asymmetric distribution identified? Second, how are these mRNAs maintained in a translationally repressed state as localization occurs? The research program proposed here aims to address these two questions. It is my hypothesis that mRNAs destined for asymmetric distribution are identified in the nucleus prior to export. Based on evidence from other model organisms and on observations in differentiated cells in mammals, it is becoming apparent that the proteins involved in RNA splicing are linked with localization. I aim to determine what components of the splicing machinery are involved in identifying mRNAs destined for localization and how they interact with components found within the cytoplasm to carry mRNA to its proper destination. During this transport, it is thought that the mRNA is kept in a silenced state in order to prevent protein production from occurring in unwanted areas. I have previously demonstrated that the RNA-binding protein and translational repressor, Pumilio 2 (Pum2) is part of the asymmetric RNA granule in neural precursor cells. I aim to determine if Pum2 and its paralogue Pum1, as well as the similar translational repressors Musashi 1 and Musashi 2, act as silencers during asymmetric RNA localization in neural precursors. Both gene families have shown evidence of acting as regulators of translation in other stem cell populations, making them ideal candidates to fulfill this role in neural precursor cells. These two central questions will be addressed using well established molecular, cellular and biochemical techniques that allow for the genetic manipulation of neural stem cells both in culture and in the intact brain. The findings will advance our understanding of asymmetric cell divisions in neural precursor cells and how this event participates in regulating the development of the brain.