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" In Vivo Proximity Labeling of Translation Initiation Complexes and Repressive RNA Granules by APEX-Seq "
Padron, Alejandro
Ingolia, Nicholas T.
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Latin Dissertation
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English
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| Record Number
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1105384
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| Doc. No
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TLpq2308191737
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| Main Entry
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Ingolia, Nicholas T.
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Padron, Alejandro
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| Title & Author
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In Vivo Proximity Labeling of Translation Initiation Complexes and Repressive RNA Granules by APEX-Seq\ Padron, AlejandroIngolia, Nicholas T.
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| College
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University of California, Berkeley
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| Date
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2019
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| student score
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2019
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| Degree
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Ph.D.
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| Page No
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83
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| Abstract
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RNA localization is a fundamentally important regulatory mechanism for the control of gene expression. I developed an in vivo subcellular RNA proximity labeling technique called APEX-Seq to monitor RNA localization and organization in the cell. In my approach, a proximity labeling enzyme is fused to a query protein and rapidly (<1 minute) biotinylates nearby RNAs in vivo upon addition of a labeling substrate, allowing their subsequent purification and analysis. We show first that APEX-Seq can distinguish the localization of transcripts to different regions of the cell. Further, we can detect the enrichment of specific transcripts in proximity to translation initiation proteins, showing that our system can capture more subtle, RNA-protein localization patterns. Finally, we present a high-resolution time course of protein and RNA condensation into stress induced RNA granules. A powerful aspect of APEX-Seq is the ability to match the spatial transcriptome with quantitative spatial proteomics, allowing for a more complete picture of the spatial landscape of the cell. Additionally, I rationally engineered a split APEX enzyme for conditional spatial proteomics and transcriptomics. I found evidence of unique translation initiation complexes through the use of split APEX on the initiation factors eIF1A/eIF4H, and eIF4A/eIF4B. Overall, these tools open the door for comprehensive and high throughout spatial transcriptomics, and proteomics with subcellular resolution.
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Genetics
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Molecular biology
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