The CUP1 gene encodes a copper metallothionein (Cup1p), which functions in maintaining the physiological levels of copper that are essential for activities of numerous proteins. The transcription of CUP1 is activated rapidly upon exposure of cells to excess copper. Cuplp sequesters excess copper from the cellular environment, thereby minimizing the deleterious effects of the metal ion. However, complete removal of copper by excess metallothionein expression is prevented by rapid shutdown of the gene, the mechanism of which is currently unknown. We are investigating RUF5, an antisense RNA generated by the CUP1 locus, as a candidate RNA involved in downregulation of CUP1. Real time RT-PCR assays suggest that RUF5 transcript levels increase as the CUP1 is being turned off. This is consistent with the hypothesis that transcription of RUF5 leads to shut down of CUP1 through the transcriptional interference mechanism. We obtained further evidence that lower CUP1 levels correlate with increased RUF5 levels using mutant strains in which CUP1 expression is weak and delayed, suggesting a fine-tuning mechanism between CUP1 and RUF5 expression to achieve proper levels of Cuplp. We are identifying the key components that are involved in this process, and determining the role of chroma-tin remodeling-modification in the regulation of RUF5.
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