PgmNr C50: Characterization of the ubiquitin-like modifier Urm1 in the Ciliate Tetrahymena thermophila.

Authors:
J. Copeland; J. Smith


Institutes
Missouri State University, Springfield, MO.


Abstract:

Ubiquitin-Related Modifier (Urm1) is a small protein with the characteristic ubiquitin Beta-grasp fold and Carboxyl termini diglycine motif. It has been implicated in two cellular functions: conjugation to substrate proteins during oxidative stress, such as pAhp1 and in the thiolation of wobble uridines in certain cytosolic tRNAs. Both processes require activation by E1-like enzyme Uba4, which has a C-terminal rhodonase domain and demonstrates both sulfurtransferase and acetyltransferase activity. Once Urm1 is activated by Uba4, the other members of the Urm1 tRNA-thiolation pathway: Ncs6, Ncs2, Tum1, Nsf1 will help mediate the transfer of the sulfur onto the uridine. Urm1 Thiolation of tRNAs is ubiquitous to life and highly conserved. It has been linked to various cellular stress responses: heat shock, misfolded protein response and oxidative stress as well as several signal transduction pathways: notably mTOR and JNK. However, it’s been shown several times that Urm1 knockouts have minimal phenotypic consequences. Urm1 is particularly interesting because it believed to be the most ancient Ubiquitin-like Protein (Ubl), and appears to be an evolutionary link between Eukaryotic Ubiquitination and Molydopterin Cofactor (MoCo) synthesis, which is present in Prokarea and most of Eukarya. This is supported by evidence that the Uba4 homolog in Arabidopsis thaliana is responsible for activating both Urm1 and MoCo Synthesis Pathways. Urm1 was first characterized in Saccharomyces cerevisiae, but there is a need to demonstrate tRNA thiolation activity of homologs in other organisms. This work will characterize Urm1 in Tetrahymena thermophila, a model organism useful for its quick growth as well as its relatively easy genetic engineering through homologous recombination. There will be bioinformatics data focusing on protein structure and function, as well as sequence homology to support the role of Urm1 in ubiquitin evolution. Expression profiles of Urm1 will be performed using qRT-PCR of T. thermophila grown in different growth conditions, focusing particularly on oxidative stress and heat shock. Fluorescence microscopy co-localization studies will be performed for Urm1 and the other pathway members, using genes that have been endogenously tagged with fluorophores.