Discovery, Characterization and Targeting of Fungal Self-splicing Introns
Date of Award
Spring 2024
Document Type
Dissertation
Degree Name
Doctor of Philosophy (PhD)
Department
Molecular, Cellular, and Developmental Biology
First Advisor
Pyle, Anna Marie
Abstract
Self-splicing introns are ribozymes (RNA enzymes) capable of catalyzing their own excision from precursor mRNA transcripts. Self-splicing introns are further divided into two structurally and mechanistically distinct classes, namely group I and group II introns. Both classes fold into compact tertiary structures, form intricate catalytic active sites and are dynamic during the stages of splicing. In light of these mechanistic features, self-splicing introns are ideal model systems to study the general principles of RNA folding, catalysis and splicing.More importantly, abundance of self-splicing introns within housekeeping genes is a prominent hallmark of fungal mitochondrial genomes. A prior proof-of-concept study has established the targetability of fungal group II introns with specific small-molecule inhibitors. Hence, self-splicing introns bear promise as a novel and attractive class of antifungal drug targets. However, the intron landscape in the mitochondrial genomes of human pathogenic fungi has been largely invisible. Furthermore, the targetability of fungal group I introns is less well understood and there is a lack of specific group I intron splicing inhibitor chemotypes. These gaps in our knowledge and toolbox have constrained further development of the intron-targeting program into a general antifungal development strategy. To bridge the gaps and to further extend the intron-targeting program, I started out by developing a bioinformatic workflow for rapid identification of self-splicing introns within the mitochondrial genomes of human pathogenic fungi, which led to the discovery of self-splicing introns in nearly all medically relevant pathogenic fungi (Chapter 2). The workflow is also capable of annotating the mitochondrial genomes in order to shed light on the intron landscapes in pathogens and predict the secondary structures of newly uncovered self-splicing introns. Guided by the predicted secondary structures, I then established a structure-first approach for prioritizing novel self-splicing introns for in vitro biochemical characterization (Chapter 3). This approach has led to the discovery of hyper-active group II introns in dimorphic fungi (Histoplasma capsulatum, Coccidioides immitis) that efficiently catalyze the self-splicing reaction, giving a rate that outperforms all known group II introns. Moreover, I have unveiled an abundance of group I introns in a variety of human pathogenic fungi spanning all three phyla in the fungal kingdom. A structurally unique IA1 lineage of introns ubiquitous in ascomycetes has been characterized extensively. The robust biochemical activity and unprecedented rapid catalytic turnover of a representative IA1 intron from Candida albicans has shed light on potential reasons accounting for its retention across species. Moreover, I have also examined the in vitro enzymological behaviors of representative IB and ID lineages of introns in Candida auris, a multi-drug resistant fungi posing an immediate threat to human health. The discovery and characterization of the self-splicing introns in medically important fungi have further enabled target-centric high-throughput screening for the development of novel intron splicing inhibitors (Chapter 4). I first developed a versatile high-throughput screening platform using the fluorescent molecular beacon assay that directly tracks the accumulation of splicing products. I then proceeded with a large screening campaign using a combination of bioactive, diversity and focused libraries to target the unique IA intron in Candida albicans. I identified multiple potent in vitro splicing inhibitors and further elucidated the binding mode of one such de novo inhibitor to the intron RNA using cryogenic electron microscope to atomic resolution. This part of work not only yielded multiple chemotypes for future development of novel antifungal agents, but also provided unprecedented insights into the molecular recognition between small molecule ligand and RNA, further pushing the boundary of the RNA-targeting field. This dissertation therefore reveals the vast potential of fungal intron targeting, establishes essential toolboxes for molecular discovery, and lays a solid foundation for the rational design and development of next-generation antifungal therapeutics.
Recommended Citation
Liu, Tianshuo, "Discovery, Characterization and Targeting of Fungal Self-splicing Introns" (2024). Yale Graduate School of Arts and Sciences Dissertations. 1331.
https://elischolar.library.yale.edu/gsas_dissertations/1331
