Nuclear translocation of Pus7 in Candida albicans

Candida albicans, an opportunistic pathogen, can cause invasive infections among hospitalized patients and the immunocompromised. C. albicans antifungal drug resistance is increasing making it the 4th most isolated organism in blood stream infections. Pseudouridine is the most prevalent RNA modification. Pseudouridine Synthase 7 (Pus7), a pseudouridine synthase cleaves the C-N bond of uridine and makes the new C-C bond on the C5 forming pseudouridine which is thought to stabilize the structure and allow it to form biochemical structures distinct from uridine. The role of Pus7 has been characterized in pseudouridylation Candida species and Saccharomyces cerevisiae with previous studies finding hundreds of pseudouridine sites among its mRNAs in the latter. In S. cerevisiae we found that Pus7 can enter the nucleus during times of stress. It is unclear if Pus7 can nuclear translocate in C. albicans. We hypothesize that Pus7 will translocate in the nucleus of C. albicans. To test this hypothesis, we designed a Pus7-GFP in a plasmid with a NAT resistance gene as a selective marker. The Pus7-GFP tagged plasmid was digested to release the fragment containing the labeled protein and marker then transformed into C. albicans. The digestion of the plasmid was successful showing two bands when visualized on an agarose gel. Initial transformations into C. albicans were unsuccessful as we observed growth of both the positive and negative transformants on 2x NAT-YPD plates. Troubleshooting of the transformations into C. albicans is still in progress.