Exposing the Functional Role of Novel Protein-RNA Interactions in PRC1 During Development

揭示 PRC1 开发过程中新型蛋白质-RNA 相互作用的功能作用

基本信息

批准号：
10766173
负责人：
Julia Tasca
金额：
$ 4.77万
依托单位：
UNIVERSITY OF PENNSYLVANIA
依托单位国家：
美国
项目类别：
财政年份：
2022
资助国家：
美国
起止时间：
2022-09-27 至 2025-09-26
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10766173
关键词：
Acute Amino Acids Architecture Bar Codes Binding Biochemical Bioinformatics Cell Lineage Cells ChIP-seq Chemicals Chromatin Chromatin Remodeling Factor Communication Complex Couples Coupling Data Data Analyses Dedications Defect Deposition Development Embryo Embryonic Development Ensure Environment Epigenetic Process Event Gene Expression Gene Silencing Gene Targeting Genes Goals Histone H2A Human Immunoprecipitation Intellectual functioning disability Investigation Kidney Libraries Link Lysine Malignant Neoplasms Maps Mass Spectrum Analysis Mediating Methods Microcephaly Molecular Mus Mutation Nature Neurodevelopmental Disorder PRC1 Protein Peptides Physiological Polycomb Proteins RNA RNA Binding RNA Biochemistry RNA-Protein Interaction Reporting Research Resolution Role Scanning System Technology Testing Training Training Programs Ubiquitin Work cell type crosslinking and immunoprecipitation sequencing design differentiation protocol directed differentiation embryonic stem cell experience experimental study functional genomics gene repression improved in vivo inducible gene expression insight interest mutant nerve stem cell neural new technology novel pluripotency protein degradation protein function skills stem cells transcriptome sequencing

项目摘要

Project Summary The goal of this proposal is to dissect the distinct molecular roles of protein-RNA interactions between RNA and the Polycomb Repressive Complex 1 (PRC1) during neural differentiation. PRC1 is a conserved chromatin modifier that represses transcription via chemical and structural alterations of chromatin architecture. PRC1 mutations cause neurodevelopmental disorders characterized by microcephaly, intellectual disabilities, and dysmorphic body features, making it crucial to gain understanding about PRC1 function. Despite its ubiquitous expression, PRC1 can target distinct sets of genes for silencing in different cell lineages, resulting in cell-type specific expression. Though there are decades of research on Polycomb protein function, a key unanswered question is how PRC1 selects different target genes in different cell types. Mounting evidence in the field indicates that major epigenetic events are regulated by interactions between RNA and chromatin-modifying proteins, including subunits of PRC1. However, PRC1-RNA interactions remain underexplored. My preliminary data indicates that at least two PRC1 subunits, SCMH1 and RING1B bind to RNA in vivo. I will investigate the regulatory role of these protein-RNA interactions in the context of directed differentiation of mouse embryonic stem cells (ESCs) into neural progenitor cells (NPCs) with the following specific aims. In Aim 1, I will identify RNAs directly bound to SCMH1 and RING1B in pluripotent ESCs using state-of-the art photocrosslinking and sequencing approaches. I will also map protein residues required for these interactions using a new technology that couples genetically encoded peptide barcodes to a mutational library for each subunit followed by a mass spectrometry readout. In Aim 2, I will investigate the functional roles of these protein-RNA interactions during neural differentiation by using our existing RNA-binding PRC1 mutants and a degrade-and-rescue approach, leveraging my lab’s expertise in acute protein degradation. The experiments in this proposal will provide the first in-depth characterization of PRC1-RNA interactions in regulating cell fate transitions during differentiation. To achieve these aims, I have developed with my sponsor and co-sponsor a rigorous and comprehensive training program with four primary goals: 1) become an expert in mass spectrometry for protein-RNA biochemistry, 2) gain experience in cutting-edge functional genomics methods, 3) increase proficiency in bioinformatics and data analysis, 4) sharpen my research communication skills. I am confident that my choice of sponsor and co-sponsor combined with my diverse training background and the collaborative nature of my training environment will enable me to achieve my goals and the proposed research plan simultaneously.

项目概要该提案的目标是剖析 RNA 之间蛋白质-RNA 相互作用的不同分子作用神经分化过程中的 Polycomb 抑制复合体 1 (PRC1) 是一种保守的染色质。通过染色质结构的化学和结构改变来抑制转录的修饰剂。突变会导致以小头畸形、智力障碍为特征的神经发育障碍畸形的身体特征，使得了解 PRC1 的功能变得至关重要，尽管它无处不在。 PRC1 可以针对不同细胞谱系中不同的基因组进行沉默，从而产生细胞类型尽管对 Polycomb 蛋白功能进行了数十年的研究，但一个关键问题尚未得到解答。问题是 PRC1 如何在不同的细胞类型中选择不同的靶基因。表明主要的表观遗传事件受到 RNA 和染色质修饰之间的相互作用的调节然而，PRC1-RNA 相互作用仍未得到充分研究。我的初步数据表明至少有两个 PRC1 亚基 SCMH1 和 RING1B 在体内与 RNA 结合。研究这些蛋白质-RNA 相互作用在小鼠定向分化中的调节作用胚胎干细胞（ESC）转化为神经祖细胞（NPC）的目的如下。在目标 1 中，我将使用最先进的技术鉴定多能 ESC 中直接与 SCMH1 和 RING1B 结合的 RNA 我还将绘制这些相互作用所需的蛋白质残基图。使用新技术将基因编码的肽条形码与每个突变库结合起来亚基随后是质谱读数。在目标 2 中，我将研究这些蛋白质-RNA 相互作用在神经分化过程中的功能作用使用我们现有的 RNA 结合 PRC1 突变体和降解和救援方法，利用我实验室的该提案中的实验将提供首次深入的蛋白质降解专业知识。 PRC1-RNA 相互作用在分化过程中调节细胞命运转变的表征。为了实现这些目标，我与我的赞助商和共同赞助商制定了严格而全面的培训计划有四个主要目标：1) 成为蛋白质-RNA 质谱分析专家生物化学，2) 获得尖端功能基因组学方法的经验，3) 提高以下方面的熟练程度生物信息学和数据分析，4）提高我的研究沟通技巧。我相信我对赞助商和联合赞助商的选择结合了我多样化的培训背景和我的培训环境的协作性质将使我能够实现我的目标和拟议的目标同时制定研究计划。