Determining the effect of RNA binding protein phosphorylation on mRNA fate

项目摘要 局部翻译是蛋白质在不同亚细胞内聚集的保守分子机制 隔间在形态上不同的细胞如神经元中，mRNA必须经过很长的距离才能到达它们的神经元。 最终目的地。RNA结合蛋白（RBP）结合靶mRNA并协调mRNA命运，包括 运输、锚定和平移。通过结合靶mRNA，RBP支持RNA-蛋白质网络， 蛋白质-蛋白质相互作用称为信使核糖核蛋白复合物（mRNP）。虽然已经 尽管我们进行了大量研究RNA-蛋白质直接相互作用的工作，但我们不知道mRNP的成分， 可能会与限制性商业惯例相互作用来调节它们。为此，我们将研究邮政编码结合蛋白1 （ZBP 1）及其靶mRNA β-肌动蛋白，以评估RBP的磷酸化如何改变蛋白质和RNA mRNP的组成。我们假设ZBP 1磷酸化改变了β-肌动蛋白mRNP的组成， 从而引起运输、锚定和平移的动态变化。我们将调查这一假设 使用两种不同的细胞类型：成纤维细胞和神经元。首先，我们将通过结合邻近性来测试这一假设。 用MS 2 RNA标记系统进行生物素化，以捕获α-肌动蛋白mRNP的整体变化 小鼠胚胎成纤维细胞中的ZBP 1磷酸化。我们将使用成像技术验证这些发现。 为了了解肌动蛋白mRNP组成的分子效应，我们将跟踪如何运输，锚定， 和翻译的各种ZBP 1磷酸化突变体的影响，使用单细胞 显微镜和分子生物学技术。用培养的海马神经元，我们将评估 β-肌动蛋白mRNA运动和翻译的变化，以响应引入ZBP 1磷酸化突变体。这些 研究对于确定ZBP 1磷酸化如何改变β-肌动蛋白mRNP的组装将是重要的 并确定这种形式的RBP调节的分子后果。该项目将在 罗伯特·H博士的指导。阿尔伯特·爱因斯坦医学院的辛格是RNA生物学的领先专家， 单分子显微镜在辛格博士的指导下，莱蒂·努涅斯将接受多学科的 在分子和细胞生物学方面的培训结合了最先进的显微镜技术。期间 在此期间，Leti将接受培训，以促进她在学术医学特别是相关的职业生涯 疾病的细胞基础。例如，ZBP 1在mRNA定位中的作用与记忆有关。 学习和肿瘤转移。

PROJECT SUMMARY Local translation is a conserved molecular mechanism of concentrating proteins within distinct subcellular compartments. In morphologically distinct cells like neurons, mRNAs must travel great distances to reach their final destinations. RNA-binding proteins (RBPs) bind target mRNAs and coordinate mRNA fates including transport, anchoring, and translation. By binding target mRNAs, RBPs support a network of RNA-protein and protein-protein interactions called the messenger ribonucleoprotein complex (mRNP). While there has been significant work to investigate direct RNA-protein interactions, we do not know constituents of the mRNP that may interact with RBPs to regulate them. For this proposal, we will investigate the zipcode binding protein 1 (ZBP1) and its target mRNA, -actin, to evaluate how phosphorylation of RBPs alters the protein and RNA composition of the mRNP. We hypothesize that ZBP1 phosphorylation alters the β-actin mRNP composition, thereby causing dynamic changes in transport, anchoring, and translation. We will investigate this hypothesis using two distinct cell types: fibroblasts and neurons. First, we will test this hypothesis by combining proximity biotinylation with the MS2 RNA labeling system to capture global changes in the -actin mRNP in response ZBP1 phosphorylation in mouse embryonic fibroblasts. We will validate these findings using imaging techniques. To understand the molecular effects of -actin mRNP composition, we will follow how the transport, anchoring, and translation are affected by various ZBP1 phosphorylation mutants using a combination of single cell microscopy and molecular biology techniques. With cultured hippocampal neurons, we will evaluate whether the -actin mRNA motility and translation changes in response to introduced ZBP1 phosphorylation mutants. These studies will be important for determining how assembly of the -actin mRNP is altered by ZBP1 phosphorylation and identify the molecular consequences of this form of RBP regulation. This project will be completed under the mentorship of Dr. Robert H. Singer at Albert Einstein College of Medicine, a leading expert in RNA biology and single molecule microscopy. Under the advisement of Dr. Singer, Leti Nunez will receive a multi-disciplinary training in molecular and cellular biology combined with expertise in state-of-the-art microscopy. During the duration of this award, Leti will receive training to advance her career in academic medicine particularly relevant to the cellular basis of disease. For example, ZBP1’s role in mRNA localization has been implicated in memory and learning and tumor metastasis.