The localization and translation of messenger ribonucleic acids (mRNAs) play crucial roles in cellular function and diseases, and are regulated by numerous RNA-binding proteins (RBPs) and small non-coding RNAs, called trans-acting factors. Biochemical and imaging methods used to study RNA interactions with these trans-acting elements have made important discoveries in characterizing how these factors regulate gene expression and determining the RNA sequence to which they bind. However, the spatiotemporal information regarding these interactions in subcellular compartments have been difficult to determine or to quantify accurately. To image and quantify native RNA and RNA–protein interactions simultaneously in situ, we developed a proximity ligation assay that combines peptide-modified RNA imaging probes. It can detect the RNAs in live cells and the interactions at a single-interaction level. Lastly, it can produce results that are easily quantifiable. We tested the specificity and sensitivity of this technique using two models: interactions between the genomic RNA and the N protein of human respiratory syncytial virus as well as those between exogenous transcripts with or without the Human antigen R (HuR) binding site and HuR. To validate this method, its accuracy and utility have been demonstrated in three models: poly(A)+ or β-actin mRNAs binding to different cytoskeleton for localization, poly(A)+ or β-actin mRNAs interacting with HuR for stabilization, and programmed cell death 4 (PDCD4) mRNA binding to HuR or T-cell intracellular antigen (TIA1) for translational regulation.
| Identifer | oai:union.ndltd.org:GATECH/oai:smartech.gatech.edu:1853/54278 |
| Date | 07 January 2016 |
| Creators | Jung, Jeenah |
| Contributors | Santangelo, Philip J. |
| Publisher | Georgia Institute of Technology |
| Source Sets | Georgia Tech Electronic Thesis and Dissertation Archive |
| Detected Language | English |
| Type | Dissertation |
| Format | application/pdf |
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