Jeenah Jung
Ph.D. Thesis Defense Presentation

Wednesday, September 3, 2014
2:00 PM
Whitaker 3115
Georgia Institute of Technology

Committee:
Philip J Santangelo, Ph.D.
Ravi V Bellamkonda, Ph.D.
Wilbur A Lam, M.D., Ph.D.
John F McDonald, Ph.D.
Gary J Bassell, Ph.D.

DEVELOPMENT OF OPTICAL IMAGING METHOD FOR DETECTING RNA-PROTEIN INTERACTIONS

Abstract:
The localization and translation of messenger ribonucleic acids (mRNAs) play crucial roles in cellular function and disease pathogenesis, and are regulated by numerous RNA-binding proteins (RBPs) and small non-coding ribonucleic acids (RNAs), called trans-acting factors. In recent years, biochemical and imaging methods used to study RNA interactions with these trans-acting elements have made several important discoveries in identifying these factors, 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 (PLA) that combines peptide-modified, multiply-labelled tetravalent RNA imaging probes (MTRIPs). This method enables sequence-specific imaging of native RNA with proximity ligation and rolling circle amplification (RCA). It is compatible with different fixation methods, and immunostaining can be performed in addition to the methodology. 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 model systems: 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 model systems: 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.