The liver plays a central role in cholesterol homeostasis through modulation of lipoprotein uptake and secretion, and cholesterol biosynthesis. MicroRNAs (miRNA) are small non-coding regulatory RNAs that post- transcriptionally control gene expression and have been demonstrated to regulate hepatic cholesterol metabolism. miRNAs are expressed in all cell-types and are present in plasma. Human and mouse lipoproteins transport specific miRNAs and deliver them to recipient cells, including hepatocytes. Lipoprotein delivery of miRNAs resulted in the reduction of specific target mRNA levels. Lipoprotein-associated miRNAs, miR-223 and miR-24, are found in liver and are computationally predicted to target cholesterol biosynthetic enzymes, as well as various lipoprotein receptors. In vitro studies have revealed that high-density lipoprotein (HDL)-miR-223 is transferred to hepatocytes by the scavenger receptor BI (SR-BI). Based on these preliminary findings, I hypothesize that specific circulating miRNAs control cholesterol metabolism through functional targeting within the liver. This proposal aims to determine the contribution of lipoprotein receptors, SR-BI and low-density lipoprotein receptor (LDLR), in transferring lipoprotein-bound miR-223 to the liver in vivo. To determine the transfer pathway, LDL-miRNA delivery will be differentiated from HDL-miRNA delivery in transfer efficiency and sub-cellular localization. To quantify the hepatic incorporation of extracellular miRNAs onto intracellular mRNA targeting complexes, photoativatable-ribonucleoside-enhanced crosslinking immunopreciptation will be performed with high-throughput small RNA sequencing. Hepatic miR-223 and miR-24 targeting of cholesterol biosynthetic enzymes and lipoprotein receptors will be validated by loss-of-function and overexpression studies in vitro. Validated targets will be assessed in vivo with miR-223-/-, SR-BI-/-, and LDLR-/- knockout mice. This proposal will systematically characterize lipoprotein-miRNA delivery and examine its role in hepatic gene regulation and cholesterol homeostasis.