(176ao) Roles of Regulatory Proteins in Controlling Endocytosis of siRNA-Containing Complexes

Short interfering RNAs (siRNAs) are 21-23 nucleotide double stranded RNAs that specifically knock down the expression of a target protein. siRNAs act via the RNA interference (RNAi) pathway to destroy targeted mRNAs and prevent their translation to proteins. siRNAs offer promise for the treatment of diseases caused by overexpression of proteins. In vivo, siRNAs are rapidly degraded by serum nucleases and cannot cross cell membranes due to electrostatic repulsion. As such, they must be complexed with a delivery vehicle. Delivery vehicles should be designed to avoid sequestration by the immune and blood filtration systems, maximize uptake by diseased cells, and encourage siRNA escape from the endosome to the cytoplasm to initiate RNAi. Designing for uptake and endosomal escape requires a clear understanding of the endocytosis pathways used by cells to internalize siRNA-containing complexes. Clathrin-mediated endocytosis, caveolin-mediated endocytosis, and macropinocytosis have been well studied since their discovery decades ago; however, more recently discovered pathways of flotillin-mediated, GRAF1-mediated, and ARF6-dependent endocytosis are regulated by unidentified proteins. To better understand regulation of these pathways, we generated a protein interaction map incorporating gene expression data across multiple cell types to identify common and unique regulators. We will confirm the identified proteins as key regulators of endocytosis through overexpression and analysis of siRNA accumulation and target silencing.