Spatially-resolved transcriptomic mapping in live cells using peroxidase-mediated proximity biotinylation
Name
1023627568-MIT.pdf
Description
Full printable version
Size
16.03 MB
Format
Adobe PDF
Checksum (MD5)
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Author(s)
Kaewsapsak, Pornchai
Advisor(s)
Alice Y. Ting.
Date Issued
2017
Publisher
Massachusetts Institute of Technology
Abstract
The spatial organization of RNA within cells is crucial for the regulation of a wide range of biological functions, spanning all kingdoms of life. However, a general understanding of RNA localization has been hindered by a lack of simple, high-throughput methods for mapping the transcriptomes of subcellular compartments. Here, we developed two methods, termed APEX-RIP and APEX-Seq. APEX-RIP combines peroxidase-catalyzed, spatially restricted in situ protein biotinylation with RNA-protein chemical crosslinking, while APEX-Seq utilizes peroxidase-catalyzed in situ biotinylation on RNA. We demonstrated that APEX-RIP can isolate RNAs from a variety of subcellular compartments, including the mitochondrial matrix, nucleus, bulk cytosol, and endoplasmic reticulum (ER) membrane, with higher specificity and coverage than conventional approaches. We furthermore identified candidate RNAs localized to mitochondria-ER junctions and nuclear lamina, two compartments that are recalcitrant to classical biochemical purification. Similarly, APEX-Seq can isolate RNAs from mitochondrial matrix, ER-associated RNAs, OMM-associated RNAs, and potentially other non-membrane bound compartments. We also revealed many non-coding RNA candidates at these sites. Since APEX-RIP and APEX-Seq are simple, versatile, and do not require special instrumentation, we envision their broad applications in a variety of biological contexts.
Description
Thesis: Ph. D., Massachusetts Institute of Technology, Department of Chemistry, 2017.
Cataloged from PDF version of thesis.
Includes bibliographical references.
Subjects
Chemistry.
MIT Department
Massachusetts Institute of Technology. Department of Chemistry
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