Audrey L. Atkin:Research

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Gene regulatory mechanisms in two fungal model organisms, Saccharomyces cerevisiae and Candida albicans, to study regulation of cell and developmental biology.

Regulation of normal cellular mRNA decay by the nonsense-mediated mRNA decay pathway

Photographs of double-labeled yeast cells showing Upf1p (green), DNA (blue) and yeast cells. This approach was used to show that Upf1p is primarily cytoplasmic.

Nonsense-mediated mRNA decay is a highly conserved mRNA decay pathway that serves an RNA surveillance mechanism by ridding cells of premature termination codon-containing mRNAs encoding potentially harmful truncated proteins. It also regulates the accumulation of approximately five percent of the normal cellular mRNAs in yeast (S. cerevisiae), fruit flies (Drosphila melanogaster) and humans. Further it is essential for mammalian viability. Thus nonsense-mediated mRNA decay serves a second important cellular function in normal regulation of gene expression. Our long-term goal is to understand the molecular mechanisms responsible for recognition and targeting of wild-type mRNAs for degradation by this pathway and the contributions of this pathway to gene regulation.

We showed that the normal cellular mRNA, PPR1 mRNA, is targeted for nonsense-mediated mRNA decay by a unique mechanism that depends on 1) the same cellular factors that are involved in the decay of nonsense mRNAs and 2) a specific region of the PPR1 mRNA. PPR1 encodes a transcription activator and increasing PPR1 mRNA levels by inhibiting nonsense-mediated mRNA decay in turn results in an increase in the expression of Ppr1-activated genes. From the lessons learned studying PPR1 mRNA decay, we developed a novel bioinformatics approach to identify other mRNAs regulated by the yeast nonsense-mediated mRNA decay pathway. In the process of identifying additional mRNAs that are regulated by the nonsense-mediated mRNA decay pathway, we discovered an mRNA that is protected from the nonsense-mediated mRNA decay pathway. This work was funded by a grant from the National Science Foundation (MCB-0444333).

Not all wild type mRNAs with NMD targeting signals are degraded by the NMD pathway. For example, the uORFs of GCN4 mRNA regulate translation of the protein coding region and this mRNA is protected from NMD. YAP1 mRNA and SSY5 mRNA, with a uORF and a long 3’-UTR respectively, are two additional examples of mRNAs that are protected from NMD. The mechanism responsible for protection of these mRNAs from NMD is unknown and is a focus of our current research. This work is funded by a grant from the National Science Foundation (MCB-1244247).

Regulation of Candida albicans morphogenesis by quorum sensing

Time lapse microscopy of C. albicans resting cells incubated in germ tube inducing conditions in the presence and absence of farnesol. In the presence of farnesol, germ tube formation is prevented. Instead the cells grow as yeasts.

C. albicans is an important opportunistic human pathogen. It normally resides in the gastrointestinal and genitourinary tract and to a lesser extent on the skin of most humans. However, given the opportunity, it can cause candidemia where it invades host tissues, progresses to growth of fungal masses in the kidney, heart or brain, and ultimately can cause death. Candida morphogenesis is important for development of candidemia. Patients with compromised immune systems are at high risk for candidemia. Many patients with candidemia die by the time laboratory diagnosis is made. Further, there is still substantial mortality of patients who receive antifungal treatment. Consequently there is a need for development of molecular probes that facilitate earlier clinical diagnosis and new classes of antifungal drugs.

Candida can grow vegetatively as yeast, hyphae or pseudohyphae. It posesses the ability to interconvert between these different morphological forms in response to its environment. There is a strong correlation between morphological interconversion and pathogenicity. The morphological transition is regulated, in part, by farnesol. Farnesol is synthesized by Candida and it blocks the conversion of yeast to hyphae or pseudohyphae in response to most, if not all, of the chemical and environmental morphogenesis inducers. Farnesol also acts as a virulence factor for systematic Candida infections in a mouse model, and the response to farnesol is unique to Candida because it does not block the morphological transition in the other dimorphic fungal species we have tested. We are part of a multidisciplinary team studying the role of farnesol. We are determining how Candida interprets signaling by farnesol at the level of gene regulation and expression, and then executes this regulation through changes in cell structure, dynamics and function. We are also examining the influence of farnesol on the interactions between "Candida" and the host immune system.

An understanding of how Candida responds to farnesol is important because it will reveal a whole new layer of fungal morphogenesis control that, in turn, should provide a series of new target sites for the design of antifungal drugs. Further, the specificity of farnesol for control of the Candida morphological transition suggests that the genes for farnesol response may be unique to Candida and thus these genes are candidates for molecular probes for rapid clinical diagnosis.

This work is funded by grants from the University of Nebraska Tobacco Settlement Biomedical Research Enhancement Fund and the UNL Research Foundation.