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.

项目摘要 局部翻译是一种保守的分子机制，可在不同的亚细胞内浓缩蛋白质 在形态上不同的细胞中的隔室，例如神经元，mRNA必须走很远的距离 最终目的地。 运输，锚定和翻译通过结合目标mRNA，RBPS支持RNA-蛋白网络 蛋白质蛋白相互作用称为核糖核蛋白复合物（MRNP） 研究直接直接RNA - 蛋白质相互作用的重要工作，我们不知道MRNP的宪章 可以与RBP相互作用以适应此建议。 （ZBP1）及其靶mRNA-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。 从而导致运输，锚定和翻译的动态变化。 使用两种不同的细胞类型：Fibreblasts和神经元。 使用MS2 RNA标记系统生物素化，以捕获-肌动蛋白MRNP的全局变化以响应 小鼠胚胎成纤维细胞中的ZBP1磷酸化。 为了了解-肌动蛋白MRNP组成的分子效应，我们将遵循运输，锚定的方式 和翻译受到使用单细胞组合的各种ZBP1磷酸化MUT的影响 显微镜和分子生物学技术。 肌动蛋白mRNA运动和翻译对引入的ZBP1磷酸化突变体的变化。 研究对于确定如何组装 并强调这种RBP调节的分子后果。 阿尔伯特·爱因斯坦医学院的罗伯特·H·辛格博士的指导，RNA生物学领域的主要专家 单分子显微镜。 分子和细胞生物学的培训结合了最新显微镜的专业知识。 该奖项的持续时间，莱蒂将接受培训以推进她的护理护理护理护理护理医学医学 例如，对于疾病的细胞基础，ZBP1在mRNA定位中的作用与记忆有关 以及学习和肿瘤转移。