Novel Repression Mechanisms for Metal Transport Genes in Saccharomyces cerevisiae

The transition metals iron and zinc are essential requirements for all living organisms. They participate in a variety of critical metabolic pathways, enzymatic activity, and provide structural support for countless proteins. Metal ion imbalance can be detrimental to organisms, as metal deficiency can alter many biological processes while metal excess can lead to toxicity. To alleviate these detrimental situations, the yeast Saccharomyces cerevisiae has developed tightly controlled regulatory mechanisms for the proper expression of proteins that control import, export, and storage of these essential metals. This regulation is required to ensure the efficient uptake, transport, and storage of specific metal ions during periods of metal concentration fluctuations. The work presented in the first chapter of this dissertation identifies the use of cryptic transcription as a means for regulatory control of the subtelomeric metal genes ZRT1 and FIT3. These transcripts arise from promoter units upstream of the ZRT1/FIT3 transcription unit and extend into the open reading frame (ORF). We show that these transcripts are degraded by the cytoplasmic Non-Sense Mediated Decay (NMD) Pathway and are only stabilized in the absence of the NMD Pathway; hence termed Cytoplasmically Degraded Cryptic Unstable Transcripts (CD-CUTs). We further show that these CD-CUTs act to prevent expression of ZRT1 by interfering with RNA polymerase II binding and activator binding (Zap1p) to the ZRT1 promoter unit. As we identified the use of CD-CUTs to mediate the repression of ZRT1/FIT3 during normal zinc/iron conditions, the mechanistic mode of action of these transcripts remained elusive. In an attempt to identify how a cytoplasmically degraded transcript can mediate transcription in the nucleus, we chose to study the cytoplasmic trafficking of these species. In chapter two, we show that the export of the ZRT1/FIT3 CD-CUTs is likely mediated by the SR-type mRNA export factor Hrb1p and dependent on the essential mRNA export factor Mex67p. Lastly, we identify an additional mode of regulation for the iron transport gene FIT3 by the glucose repressor Mig1p. Mig1p has been extensively characterized as a repressor of glucose responsive genes and in chapter three, we show that this regulation can be applied to iron regulation. We believe that Mig1p regulates the repression of FIT3 during conditions of normal iron and/or after switch back to normal iron conditions following iron starvation to prevent the deleterious expression of FIT3, thereby preventing the potentially harmful effects of iron toxicity