Spatiotemporally resolved proteomics, transcriptomics, and interactomics with an engineered peroxidase
Abstract/Contents
- Abstract
- Spatial compartmentation evolved as a key organizing principle of living cells to regulate molecular complexes as well as the biological processes they carry out. The subcellular localization of a biomolecule and the molecular identities of its immediate neighbors not only dictate its expression and modification, but also serve to define its biological function. Thus, a mechanistic understanding of cellular processes, whether in homeostatic or pathological states, requires elucidation of the spatial patterns of molecular localization and interaction on a system level. Traditional biochemical approaches, such as subcellular fractionation and affinity purification, have contributed greatly towards this goal. However, impurities from contaminant molecules and loss of material during these procedures greatly limit their specificity and depth of biological investigation. Fluorescence microscopy-based methods provides excellent spatial resolution, but their throughput is limited by a handful of non-overlapping fluorophores. Hence, there remains a need to develop and apply novel technologies to systematically chart molecular maps inside living cells. The work presented herein seeks to address the above issues through a novel set of chemical biology tools based on engineered peroxidases. These genetically encoded enzymes are adapted and applied for promiscuous biotinylation and high-throughput identification of endogenous biomolecules (e.g. proteins and RNAs) at specific cellular loci. Chapter 1 describes the application of the engineered ascorbate peroxidase (APEX) for proximity labeling and proteomic mapping of the human mitochondrial nucleoid complex, which houses the mitochondrial DNA. This chapter details the development of APEX labeling method for the nucleoid protein complex, covering fluorescence microscopy imaging, electron microscopy imaging, and biochemical analysis of the APEX constructs employed. Subsequent 1-minute live-cell biotinylation followed by quantitative, ratiometric proteomics enriched 37 nucleoid proteins, 7 of which had never previously been associated with the nucleoid. The specificity of the dataset is very high, and three hits were validated by orthogonal means. For one novel nucleoid-associated protein, FASTKD1, we discovered its role in regulating mitochondrial complex I via specific repression of ND3 mRNA. This study demonstrates that APEX is a powerful tool for mapping macromolecular complexes in living cells, and can identify proteins and pathways that have been missed by traditional approaches. Chapter 2 extends the spatial proteomic method from cultured cells in vitro to intact tissues in vivo. In collaboration with a graduate student, Jiefu Li, in Dr. Liqun Luo's group, we developed a cell-type-specific, spatiotemporally resolved approach to profile cell-surface proteomes in intact Drosophila brain tissues with genetically encoded horseradish peroxidase (HRP). Quantitative profiling of cell-surface proteomes of Drosophila olfactory projection neurons (PNs) in different brain development stages systematically revealed an inverse regulation of wiring molecules and synaptic molecules in the transition from developing to mature PNs. The dataset also provides a rich resource for nominating candidate players that are key to olfactory wiring during PN maturation. Follow-up genetic screen of the candidates identified 20 new regulators of neural circuit assembly at the cell surface, many of which belong to evolutionarily conserved protein families not previously linked to neural development. This chapter highlights the power of spatiotemporally resolved in situ proteomic profiling in discovering novel components of complex biological processes in vivo. Chapter 3 further expands the scope of APEX-omics from protein labeling/spatial proteomics to RNA labeling/spatial transcriptomics. This chapter details the development, validation, and application of a novel RNA sequencing method, APEX-seq, based on direct proximity labeling of RNA using the peroxidase enzyme APEX2. In collaboration with a postdoctoral researcher, Furqan Fazal, in Dr. Howard Chang's lab, we applied APEX-seq in nine distinct subcellular locations to produce a nanometer-resolution spatial map of the human transcriptome. This novel technique revealed extensive patterns of localization for both noncoding RNAs and messenger RNAs as well as their transcript isoforms. In addition, the dataset provides several biological insights and hypotheses, including a radial organization of the nuclear transcriptome gated by the inner surface of the nuclear pore. Importantly, two distinct pathways of messenger RNA localization to mitochondria was identified, each associated with specific sets of transcripts for building complementary macromolecular complexes within the organelle. APEX-seq should be widely applicable, enabling comprehensive investigations of the subcellular spatial transcriptome. Chapter 4 represents an intersection of RNA and protein biology, detailing the development of RNA-targeted APEX for mapping the interactome associated with specific cellular RNAs. We developed two complementary strategies based on APEX-catalyzed proximity biotinylation of RNA interacting proteins. Leveraging the bacteriophage MS2 system and an improved CRISPR-Cas13 system, we targeted APEX to specific cellular RNAs and performed 1-minute live cell proximity biotinylation to capture endogenous protein interaction partners. Application of these approaches to the human telomerase RNA (hTR) recovered known interactions, in addition to more than a dozen potentially novel protein interaction partners. Together with a postdoctoral researcher, Boxuan Zhao, in the lab, we validated the previously unknown interaction of hTR with the RNA N6-methyladenosine (m6A) demethylase ALKBH5 and found that ALKBH5 binding to hTR dynamically regulates its m6A modification, which is required for telomerase complex assembly and maintaining telomerase activity. These results highlight the ability of MS2 and Cas13-based APEX2 targeting to uncover novel RNA-centered protein interactions in living cells
Description
| Type of resource | text |
|---|---|
| Form | electronic resource; remote; computer; online resource |
| Extent | 1 online resource |
| Place | California |
| Place | [Stanford, California] |
| Publisher | [Stanford University] |
| Copyright date | 2020; ©2020 |
| Publication date | 2020; 2020 |
| Issuance | monographic |
| Language | English |
Creators/Contributors
| Author | Han, Shuo, 1992- |
|---|---|
| Degree supervisor | Ting, Alice Y |
| Thesis advisor | Ting, Alice Y |
| Thesis advisor | Bertozzi, Carolyn R, 1966- |
| Thesis advisor | Khosla, Chaitan, 1964- |
| Degree committee member | Bertozzi, Carolyn R, 1966- |
| Degree committee member | Khosla, Chaitan, 1964- |
| Associated with | Stanford University, Department of Chemistry. |
Subjects
| Genre | Theses |
|---|---|
| Genre | Text |
Bibliographic information
| Statement of responsibility | Shuo Han |
|---|---|
| Note | Submitted to the Department of Chemistry |
| Thesis | Thesis Ph.D. Stanford University 2020 |
| Location | electronic resource |
Access conditions
- Copyright
- © 2020 by Shuo Han
- License
- This work is licensed under a Creative Commons Attribution Non Commercial 3.0 Unported license (CC BY-NC).
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