EXPLORATION OF CELL-BASED METHODS TO DISSECT LNCRNA-PROTEIN INTERACTIONS
Johns Hopkins University
Long noncoding RNAs (lncRNAs) have been increasingly important in understanding regulations of gene expressions, thanks to the discovery of non-protein-coding genes and transcripts with the advent of high-throughput sequencing. It is known that most of the lncRNA functioning mechanisms involve interactions with RNA-binding proteins. Various in vitro experiments, such as in vitro transcribed RNA pulldown and electrophoretic mobility shift assay (EMSA), have been applied to study lncRNA-protein binding. However, the mechanisms of their interactions in the cell environment remain mysterious for most lncRNAs. Our goal is to explore cell-based methods to explore lncRNA-protein interactions. These include MPRNA-IP (Massively Parallel RNA Assay-Immunoprecipitation), a high throughput experiment developed by our lab, and CLIP-qPCR (Crosslinking Immunoprecipitation and qPCR) with RNA deletion mutants or RNase treatment. We focused on two lncRNA-protein interactions, cytosolic interaction NORAD-PUM2 and intranuclear interaction HOTAIR-EZH2. We detected a well-known PUM2-binding RNA sequence motif in NORAD, validating our methods for RNA-protein interactions. Regarding EZH2-HOTAIR interaction, our experiments showed that the EZH2 protein interacts with the 5’ end of lncRNA HOTAIR, as previous in vitro assays showed. But we unexpectedly identified that EZH2 also has a binding affinity with the middle region of HOTAIR while inside the cell nucleus. Taken together, our results present representative examples of applying multiple methods to study lncRNA-protein interactions inside the cells and provide new insights into the mechanism of lncRNA-protein interactions.
lncRNA, MPRNA-IP, CLIP-qPCR, HOTAIR, EZH2