Characterization of microRNA-Protein Interactions and Genome-Scale Screening for Modifiers of LDL Cholesterol Uptake

Abstract

MicroRNAs (miRNAs) are small noncoding RNAs that regulate protein expression via post-transcriptional silencing of target genes. These small RNAs are implicated in the regulation of nearly all biological processes, and global miR biogenesis is altered in many cancers. Additionally, miRNA-binding proteins have been shown to modulate miR biogenesis, presenting a promising avenue for targeting miRNA dysregulation in disease. Herein, high-throughput screening and proteomics-mediated approaches are used for small molecule targeting and novel characterization of miRNA–protein interactions, respectively. These works culminated in the discovery of SART3 as a pre-miR-34a–binding protein whose overexpression leads to cell cycle arrest in the G1 phase, as described in Chapter 2, as well as the establishment of the first complementation assay for high throughput screening of RNA–protein interactions, as described in Chapter 3. In a second project, regulators of cholesterol-carrying low-density lipoprotein (LDL) uptake are interrogated in liver cells. Cardiovascular diseases are the leading cause of death in the United States, and high LDL cholesterol is a major risk factor for atherosclerosis. Mechanisms outside of LDL receptor-mediated endocytosis are poorly understood; to address this gap, a series of functional genomic CRISPR screens are conducted to identify genetic modulators of LDL uptake in hepatocytes. As described in Chapter 5, these studies identified ~150 previously unrecognized genes with putative roles in LDL uptake. Additionally, secondary screens for transferrin uptake, LDL receptor abundance, and LDL uptake in HepG2 cells and LDLR-deleted cells gave mechanistic insights related to the specificity, generalizability, and LDLR-dependence of these gene candidates. Furthermore, these results highlighted the statistical power of using focused secondary libraries with greater depth to follow up on genome-wide screens.