Date of Award
8-2026
Document Type
Dissertation
Degree Name
Doctor of Philosophy (PhD)
Department
Genetics and Biochemistry
Committee Chair/Advisor
Andrei Alexandorv
Committee Member
Hong Luo
Committee Member
James Morris
Committee Member
Jennifer Mason
Abstract
A number of human diseases arise from dysregulation of non-coding RNAs, whose biogenesis, processing, and degradation depend on nuclear pathways composed of proteins, RNAs, and ribonucleoproteins (RNP). However, not all components of these pathways have been discovered, in part because the nuclear localization of these pathways precluded their identification by high-throughput forward genetics. We developed forward genetic strategies to overcome this barrier, which enabled identification of previously unrecognized components of human nuclear RNA pathways. First, we developed the Mirror approach, a single-cell platform that mirrors the activity of nuclear pathways acting on the 3′ end of human long non-coding RNA MALAT1 with cytoplasmic fluorescence. Using this approach, we identified nearly all components of RNase P, the RNA Exosome, and the Nuclear Exosome Targeting (NEXT) complex, as well as two additional factors BRF2 and DDX59. We further found that the DEAD-box helicase DDX59 is required for minor intron splicing, providing the mechanism for its association with the rare human genetic disorder, oral-facial-digital (OFD) syndrome. Second, in a separate genome-wide forward genetic screen to identify components of human RNase MRP, an RNP enzyme that processes pre-rRNA at ITS1 site 2, we identified two poorly characterized human genes, which we named RMP24 and RMP64. We further demonstrated that RMP24 and RMP64 represent previously unknown essential human RNase MRP-specific protein subunits, defining the composition of the human RNase MRP. Together, our work established a forward genetic platform for identifying human pathways acting on strictly nuclear non-coding RNAs and identified previously unknown components of human pathways.
Recommended Citation
Che, Rui, "Forward Genetic Identification of Human Nuclear Non-Coding RNA Pathways" (2026). All Dissertations. 4373.
https://open.clemson.edu/all_dissertations/4373