Functions of RNA exosome and tRNA splicing ligase in Mendelian and Infectious diseases

Khondakar Sayef Ahammed, MS (Advisor: Ambro van Hoof, PhD)

RNA maturation and degradation reactions are important for health and survival of all organisms. In humans, defects in these processes cause various mendelian diseases, while drugs that inhibit pathogen-specific RNA processing enzymes promise to be effective treatment options for infectious diseases. The RNA exosome complex is a major 3’ RNase in eukaryotes that catalyzes the processing and degradation of a wide range of substrates in the nucleus and cytoplasm. Single amino acid mutations in RNA exosome subunits cause a range of Mendelian diseases collectively referred to as exosomopathies. However, distinguishing these disease-causing variants from non-pathogenic ones remains challenging, and the mechanism by which these variants cause disease is largely unknown.

Here, I develop a hybrid yeast/mammalian RNA exosome model of exosomopathies by systematically replacing the individual yeast subunits with their corresponding human or mouse orthologs. This allows us to unambiguously assess the damaging effects of the exact patient variant in budding yeast. Functional analysis of the disease-associated variants utilizing this genetic tool revealed defects in RNA exosome function caused by previously known as well as uncharacterized variants in several replaceable subunits, including EXOSC1, EXOSC2, EXOSC4, EXOSC7, and EXOSC9. Further detailed investigation of the two damaging EXOSC1 variants, using orthologous mutations in the corresponding yeast subunit Csl4, reveals malfunction in the nuclear and cytoplasmic functions of RNA exosome. These two patient-derived mutations are located in functionally redundant domains of Csl4, and each variant partially impairs RNA exosome function by disrupting its corresponding domain. Genetic and transcriptome analysis of these csl4 variants implies the N- and C-terminal domains of Cs4 have distinct and overlapping in vivo functions. Furthermore, these Csl4 domains maintain a ‘bipartite functional interaction’ with nuclear cofactors (Rrp6, Mpp6, and Mtr4), where one of the interactions is required for the essential function of the exosome. Thus, analyzing disease variants in the budding yeast model provides important insights into RNA exosome defects caused by patient-derived variants and elucidates the subunit-specific role of Csl4/EXOSC1 in nuclear RNA exosome function.

Another aspect of my thesis was dedicated to understanding the function of fungal tRNA ligase that is involved in the processing of intron-containing pre-tRNAs. The first step of the pre-tRNA splicing mechanism is conserved and involves cleavage by tRNA splicing endonuclease (TSEN), resulting in 5’ exon, 3’ exon, and intron fragments. Fungi use a 'heal and seal' pathway that requires three distinct catalytic domains of the tRNA ligase enzyme, Trl1. In contrast, humans use a 'direct ligation' pathway carried out by RTCB, an enzyme completely unrelated to Trl1. Because of these mechanistic differences, Trl1 has been proposed as a promising drug target for fungal infections.

In this study, I systematically tested whether Trl1 meets the key criteria for a viable antifungal drug target candidate. To validate Trl1 as a broad-spectrum drug target, I show that fungi from three different phyla contain Trl1 orthologs with all three catalytic domains. This includes the major invasive human fungal pathogens, and these proteins can each functionally replace yeast Trl1. In contrast, species from the order Mucorales, including the pathogens Rhizopus arrhizus and Mucor circinelloides, have an atypical Trl1 that contains the sealing domain but lacks both healing domains. These sealing-only Trl1 orthologs can functionally complement defects in the corresponding domain of yeast Trl1 and use a conserved catalytic lysine residue. Thus, Mucorales use a sealing-only enzyme together with unidentified non-orthologous healing enzymes for their heal and seal pathway. Functional investigation of the individual domains of Trl1 in pathogenic fungi species reveals that the ligase domain is the domain to target for antifungal development.

One of the key challenges in developing Trl1 inhibitors is the lack of a robust, sensitive, and rapid in vivo drug screening assay system for screening Trl1 inhibitors. To enable the discovery of Trl1 inhibitors, I designed a cell-based assay system that can be used in a high-throughput setting to identify compounds that specifically inhibit fungal Trl1. Furthermore, while validating Trl1 as a potential drug target, I uncovered several species-specific biological aspects of fungal Trl1, which provide new insights into our understanding of the tRNA splicing pathway in fungi.

Advisory Committee:

• Ambro van Hoof, PhD, Chair
• Swathi Arur, PhD
• Nicholas De Lay, PhD
• Michael Lorenz, PhD
• William Margolin, PhD
• Kevin Morano, PhD