Determining the effect of RNA binding protein phosphorylation on mRNA fate

确定 RNA 结合蛋白磷酸化对 mRNA 命运的影响

• 批准号: 10477945
• 负责人: Leti Nunez
• 金额: $ 4.66万
• 依托单位: ALBERT EINSTEIN COLLEGE OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-09-01 至 2024-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10477945
• 关键词: Actins; Address; Affect; Affinity; Award; Binding; Binding Proteins; Biology; Biotinylation; Capsid Proteins; Cell Line; Cell physiology; Cells; Characteristics; Complex; Data; Defect; Destinations; Detection; Diffuse; Disease; Distal; Embryo; Event; Fibroblasts; Global Change; Goals; Hippocampus (Brain); Image; Imaging Techniques; Investigation; Kinetics; Knowledge; Label; Learning; Manuscripts; Measures; Medicine; Memory; Mentorship; Messenger RNA; Methodology; Microscopy; Modeling; Molecular; Molecular Biology Techniques; Molecular and Cellular Biology; Morphology; Mus; Neoplasm Metastasis; Neurons; Pattern; Peroxidases; Phosphorylation; Physiological; Preparation; Process; Proteins; Proteomics; RNA; RNA-Binding Proteins; RNA-Protein Interaction; Regulation; Reporter; Resolution; Role; Site; Spatial Distribution; Stimulus; Synaptic plasticity; System; Testing; Training; Translations; Travel; Tyrosine; Work; ascorbate; beta Actin; career; cell motility; cell type; college; genetic regulatory protein; imaging approach; insight; knock-down; mRNA tagging; messenger ribonucleoprotein; migration; molecular imaging; multidisciplinary; mutant; protein protein interaction; response; single molecule; small hairpin RNA; spatiotemporal; stem; trafficking; tumor

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.

项目概要 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 隔间。在神经元等形态不同的细胞中，mRNA 必须经过很长的距离才能到达它们的位置。 最终目的地。 RNA 结合蛋白 (RBP) 结合靶标 mRNA 并协调 mRNA 的命运，包括 运输、锚定和翻译。通过结合靶标 mRNA，RBP 支持 RNA 蛋白和 蛋白质与蛋白质之间的相互作用称为信使核糖核蛋白复合物（mRNP）。虽然已经有 虽然研究 RNA-蛋白质直接相互作用的重要工作，但我们不知道 mRNA 的成分 可能与 RBP 相互作用来调节它们。对于本提案，我们将研究邮政编码结合蛋白 1 (ZBP1) 及其靶标 mRNA、-肌动蛋白，以评估 RBP 磷酸化如何改变蛋白质和 RNA mRNP 的组成。我们假设 ZBP1 磷酸化改变了 β-肌动蛋白 mRNP 组成， 从而引起运输、锚定和平移的动态变化。我们将研究这个假设 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度来检验这个假设 使用 MS2 RNA 标记系统进行生物素化，以捕获响应中 -肌动蛋白 mRNA 的整体变化 小鼠胚胎成纤维细胞中的 ZBP1 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，我们将了解转运、锚定、 和翻译受到使用单细胞组合的各种 ZBP1 磷酸化突变体的影响 显微镜和分子生物学技术。通过培养的海马神经元，我们将评估是否 -肌动蛋白 mRNA 运动和翻译变化响应引入的 ZBP1 磷酸化突变体。这些 研究对于确定 ZBP1 磷酸化如何改变 -肌动蛋白 mRNP 的组装非常重要 并确定这种 RBP 调节形式的分子后果。该项目将在 阿尔伯特·爱因斯坦医学院 Robert H. Singer 博士的指导，他是 RNA 生物学领域的领先专家 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的 分子和细胞生物学培训与最先进的显微镜专业知识相结合。期间 在此奖项期间，莱蒂将接受培训，以推进她在学术医学领域的职业生涯，尤其相关 疾病的细胞基础。例如，ZBP1 在 mRNA 定位中的作用与记忆有关 以及学习和肿瘤转移。