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

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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.