Mechanistic studies of RNA-binding proteins regulating local RNA translation at synapses

神经元是两极分化的细胞，有许多树突和从细胞体延伸出来的轴突。虽然大部分神经元蛋白质合成发生在其躯体间，但局部蛋白质合成也发生在细胞的远端区域。需要在神经元的特定位置调节蛋白质合成是一个后勤障碍，必须克服，以保持突触的可塑性和正常的神经功能。为了满足神经元内局部翻译的需求，mRNAs被RNA结合蛋白(RBPs)结合，形成针对特定局部翻译位点的核糖核蛋白(RNP)复合体。我们假设，一些限制性商业惯例在调节细胞内信号转导途径的局部翻译方面具有不可或缺的作用。因此，我们的研究计划的重点是确定限制性商业惯例本身是如何调控的，以及它们如何控制突触的局部mRNA翻译。虽然局部翻译发生在神经元的突触前和突触后，但关于局部翻译的调节机制和限制性商业惯例在这一过程中的作用仍然存在许多问题。我们的计划重点是了解限制性商业惯例，如融合在肉瘤(FUS)，如何控制本地翻译的机制。在研究哺乳动物雷帕霉素(MTOR)信号通路的靶点时，我们观察到mTOR激酶的药物抑制刺激了FUS与多聚体的结合，多聚体是将mRNA翻译成蛋白质的分子复合体。我们进一步研究了FUS与多聚体的功能联系，发现它与停滞的多聚体有关。与这一效应一致的是，当我们阻止原代神经元培养中的神经元活动时，我们通过超分辨率成像观察到FUS与核糖体亚基更多的共定位。综上所述，我们的数据表明，FUS受到细胞内信号通路的调节，这些信号通路决定了FUS在调节突触局部翻译中的作用。在这里，我们的计划旨在确定细胞信号通路如何调节FUS来控制突触的局部翻译。这项资助的目的是：1)确定影响翻译的细胞信号通路如何调节突触上的FUS活性；2)确定FUS的翻译后调节以及对局部翻译的影响；以及3)确定突触多聚体中FUS蛋白-蛋白质和蛋白质-RNA的相互作用对翻译抑制或激活的反应。这项提议的发现将作为迈向我们研究计划长期目标的垫脚石，该计划旨在确定局部翻译如何在神经元的生命周期内控制突触可塑性。我们希望我们提出的研究将为控制局部蛋白质表达的调控机制提供新的见解，并导致开发控制局部翻译的新方法。

Neurons are polarized cells that have numerous dendritic processes and an axon that extend away from the cell body. While the majority of neuronal protein synthesis occurs in its somatic compartment, local protein synthesis also occurs in distal regions of the cell. The requirement for regulated protein synthesis at specific locations in neurons is a logistical hurtle that must be overcome to maintain synaptic plasticity and proper neurological functions. To meet the demands of local translation within neurons, mRNAs are bound by RNA binding proteins (RBPs) forming ribonucleoprotein (RNP) complexes that are targeted to specific sites of local translation. We hypothesize that some RBPs have an integral role in regulating local translation in response to intracellular signal transduction pathways. Therefore, the focus of our research program is to determine how RBPs are themselves regulated and how they control local mRNA translation at synapses. While local translation occurs in both pre- and post-synapses of neurons, many questions still remain regarding the regulatory mechanisms of local translation and the role of RBPs in this process. Our program is focused on understanding the mechanisms of how RBPs, like Fused in Sarcoma (FUS), control local translation. While investigating the mammalian target of rapamycin (mTOR) signaling pathway, one of the major signaling pathways that regulates mRNA translation, we observed that pharmacological inhibition of mTOR kinase stimulates the association of FUS with polysomes, the molecular complex that translates mRNA into protein. We have further examined the functional association of FUS with polysomes and found that it associates with stalled polysomes. Consistent with this effect, when we blocked neuronal activity in primary neuron cultures, we observed more co-localization of FUS with ribosomal subunits by super resolution imaging. Taken together, our data suggest that FUS is regulated by intracellular signaling pathways that determine its role in regulating local translation at the synapse. Herein, our program aims to determine how cell signaling pathways regulate FUS to control local translation at synapses. This grant aims to: 1) Determine how cell signaling pathways, which impact translation, regulate FUS activity at the synapse; 2) Determine the post-translational regulation of FUS and the impact on local translation; and 3) Identify the FUS protein-protein and protein-RNA interactions in synaptic polysomes in response to translation inhibition or activation. Findings from this proposal will act as a stepping stone towards the long term goal of our research program, which is to determine how local translation controls synaptic plasticity over the life-time of a neuron. We hope our proposed studies will provide novel insights into the regulatory mechanisms that control local protein expression and lead to the development of new ways to control local translation.