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

神经元是极化细胞，具有许多树突状突起和从细胞体延伸的轴突。虽然大多数神经元蛋白质合成发生在其体细胞区室中，但局部蛋白质合成也发生在细胞的远端区域。在神经元中特定位置调节蛋白质合成的要求是必须克服的逻辑障碍，以维持突触可塑性和适当的神经功能。为了满足神经元内局部翻译的需求，mRNA被RNA结合蛋白（RBP）结合，形成靶向局部翻译特定位点的核糖核蛋白（RNP）复合物。我们推测，一些RBPs在调节细胞内信号转导途径的本地翻译中起着不可或缺的作用。因此，我们研究计划的重点是确定RBP本身是如何调节的，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以及RBP在这一过程中的作用仍存在许多问题。我们的计划专注于了解RBP如何控制局部翻译的机制，如融合肉瘤（FUS）。在研究哺乳动物雷帕霉素靶蛋白（mTOR）信号通路（调节mRNA翻译的主要信号通路之一）时，我们观察到mTOR激酶的药理学抑制刺激FUS与多聚体（将mRNA翻译为蛋白质的分子复合物）的结合。我们进一步研究了FUS与多聚核糖体的功能相关性，发现它与停滞的多聚核糖体相关。与这种效应一致，当我们阻断原代神经元培养物中的神经元活性时，我们通过超分辨率成像观察到更多的FUS与核糖体亚基的共定位。两者合计，我们的数据表明，FUS是由细胞内信号通路，决定其在调节突触的本地翻译的作用。在此，我们的计划旨在确定细胞信号通路如何调节FUS以控制突触的局部翻译。这笔赠款的目的是：1）确定影响翻译的细胞信号传导途径如何调节突触处的FUS活性; 2）确定FUS的翻译后调节和对局部翻译的影响;以及3）鉴定响应于翻译抑制或激活的突触多聚核糖体中的FUS蛋白-蛋白和蛋白-RNA相互作用。 这项提案的发现将作为我们研究计划长期目标的垫脚石，该目标是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，并导致控制局部翻译的新方法的发展。

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.