Determining the effect of RNA binding protein phosphorylation on mRNA fate

项目总结 局部翻译是一种将蛋白质集中在不同亚细胞内的保守分子机制 车厢。在像神经元这样形态截然不同的细胞中，mRNAs必须经过很长的距离才能到达它们的 最终目的地。RNA结合蛋白(RBPs)结合靶向mRNAs并协调mRNAs的命运，包括 运输、锚定和翻译。通过结合靶mRNAs，限制性商业惯例支持RNA-蛋白质网络，并 蛋白质之间的相互作用称为信使核糖核蛋白复合体(MRNP)。虽然已经有了 在研究直接RNA-蛋白质相互作用方面的重要工作，我们尚不知道mRNP的成分 可能会与限制性商业惯例相互作用，以对其进行监管。对于这项提议，我们将研究邮政编码结合蛋白1 (ZBP1)及其靶基因-肌动蛋白，以评估限制性商业惯例的磷酸化如何改变蛋白质和核糖核酸 MRNP的组成。我们假设ZBP1的磷酸化改变了β-肌动蛋白mRNP的组成， 从而导致运输、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，我们将通过组合邻近度来检验这一假设 利用MS2RNA标记系统进行生物素化以捕捉-肌动蛋白mRNP的全球变化 小鼠胚胎成纤维细胞中ZBP1的磷酸化。我们将使用成像技术来验证这些发现。 为了了解-肌动蛋白mRNP组成的分子效应，我们将遵循如何运输、锚定、 和翻译受到各种ZBP1磷酸化突变体使用单细胞组合的影响 显微镜和分子生物学技术。用培养的海马神经元，我们将评估是否 -肌动蛋白mRNA在引入ZBP1磷酸化突变体后的运动和翻译变化。这些 研究对于确定zbp1磷酸化如何改变-肌动蛋白mrnp的组装具有重要意义。 并确定这种形式的RBP调控的分子后果。这项工程将在 指导阿尔伯特·爱因斯坦医学院的罗伯特·H·辛格博士，他是RNA生物学和 单分子显微镜。在辛格博士的建议下，莱蒂·努涅斯将接受多学科 分子和细胞生物学方面的培训与最先进的显微镜技术相结合。在.期间 这一奖项的期限，Leti将接受培训，以推进她的职业生涯特别相关的学术医学 疾病的细胞基础。例如，ZBP1的S在基因定位中的作用被认为与记忆有关 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.