Identifying dsRNA binding proteins in human cell lysate through dsRNA pulldown assay

Abstract

dsRNA-binding proteins (dsRBPs) specifically bind to double-stranded RNA (dsRNA) and their interaction plays an important role in cellular immune response and metabolism. However, the study of dsRBPs is still insufficient, and the method to accurately classify which is dsRBPs is not yet verified. In this thesis, I showed that pulldown assay is an appropriate method to collect dsRBPs from whole cell lysate. Among the various conditions, I found the optimal conditions to identify dsRBPs within cells and verified them from the experiments. From this optimized experiment, I successfully isolated only dsRBPS from human cell lysates. To identify these dsRBP lysates, protein aggregation should be avoided. Therefore, I tested an experimental model that uses RNase A to degrade dsRNA and consequently elute the protein. Additionally, after separating cells into cytoplasm and nucleus by cell fractionation, I test whether dsRBPs in each lysate could be extracted by pull-down analysis. Although additional research is needed, my study presents an experimental model that can be widely applied in future dsRNA-binding protein screening experiments.