Import of double-stranded RNA and cholesterol into cells in C. elegans - PROJECT SUMMARY RNAi inference (RNAi) is a mechanism by which double-stranded RNA (dsRNA) of a specific sequence is used to prevent the expression of a gene with matching sequences. This process has been successfully harnessed for clinical applications that allow for the repression of disease-causing genes, including those related to cardiovascular diseases. Despite the clinical significance of this mechanism, gaps in knowledge surrounding dsRNA transport into cells remain. Currently approved dsRNA-based therapeutics are delivered through endocytosis and subsequently require a transporter to enter the cytoplasm. This cytoplasmic entry occurs at low rates in mammals through unclear mechanisms, placing limits on targeted dsRNA delivery. The nematode C. elegans efficiently transports dsRNA capable of gene silencing between cells using the transmembrane protein SID-1. SID-1 has one paralog known to transport cholesterol and two others with unclear functions, in addition to two structurally conserved mammalian homologs implicated in both processes. Recent bioinformatic analyses within the Jose lab have identified a new homolog of the human Niemann Pick cholesterol transporter NPC2 in C. elegans. This cholesterol transporter homolog has been reported to interact with SID-1, and transcriptome analyses indicate that it responds at the RNA level to disruptions in both cholesterol homeostasis and SID-1- based dsRNA transport. Understanding what conserved transporter features may facilitate or block dsRNA entry to cells could shed light on the mechanism by which SID-1 family proteins operate, which remains unclear. Understanding how dsRNA transport pathways interact with cholesterol regulatory pathways could provide insights to dsRNA therapeutic design, particularly for those targeting diseases associated with disrupted cholesterol homeostasis. Therefore, we hypothesize that (1) based on structural homology, the two uncharacterized SID-1 paralogs may play a role in dsRNA import and (2) dsRNA transport pathways intersect with cholesterol transport pathways through interaction between SID-1 and our newly identified Niemann-Pick cholesterol transporter. The objective of this project is to investigate these hypotheses through a combination of genetic knockouts of candidate proteins, biochemical analyses of hypothesized interactions and RNAi pathway intermediates, and joint visualization of transporters and metabolites. I will carry out these inquiries within the Jose, lab, which has an extensive track record of studying RNA biology and gene silencing. In addition to the broader significance of this work, pursuing these research questions will provide me with the necessary training to pursue a future career as an independent investigator. My progress toward this goal within the Jose lab is guided by an Individual Development Plan, which outlines targeted and actionable areas for improvement and provides a framework of training which will facilitate the proposed research.
