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

项目摘要 这个提议的目的是剖析RNA之间蛋白质-RNA相互作用的不同分子作用， 和多梳抑制复合物1（PRC 1）在神经分化过程中的作用。PRC 1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符。PRC1 突变导致以小头畸形、智力残疾和 畸形的身体特征，这使得了解PRC 1功能至关重要。尽管它无处不在 表达，PRC 1可以靶向不同的基因组，在不同的细胞谱系中沉默，导致细胞类型 具体表达。虽然对Polycomb蛋白功能的研究已经有几十年了，但一个关键的问题没有得到解决。 问题是PRC 1如何在不同的细胞类型中选择不同的靶基因。实地收集证据 表明主要的表观遗传事件是由RNA和染色质修饰之间的相互作用调节的， 蛋白质，包括PRC 1的亚基。然而，PRC 1-RNA相互作用仍有待研究。 我的初步数据表明，至少有两个PRC 1亚基，SCMH 1和RING 1B在体内与RNA结合。我会 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），具体目的如下。 在目标1中，我将使用最先进的方法鉴定多能ESCs中与SCMH 1和RING 1B直接结合的RNA。 光交联和测序方法。我还将绘制这些相互作用所需的蛋白质残基 使用一种新技术，将基因编码的肽条形码与突变文库偶联， 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， 利用我们现有的RNA结合PRC 1突变体和降解和拯救方法，利用我的实验室的 急性蛋白质降解的专业知识。本提案中的实验将提供第一个深入的 PRC 1-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一个严格而全面的 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 生物信息学和数据分析，4）提高我的研究沟通技巧。 我相信，我对赞助商和共同赞助商的选择，加上我多样化的培训背景， 我的培训环境的协作性质将使我能够实现我的目标和建议 研究计划同时进行。

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.