Browsing Theses and Dissertations School of Pharmacy by Title "A Tale of Two Zinc Fingers: Structure and Functional Studies of CCCH type Zinc Finger Proteins CPSF30 and TTP involved in RNA Regulation"
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A Tale of Two Zinc Fingers: Structure and Functional Studies of CCCH type Zinc Finger Proteins CPSF30 and TTP involved in RNA RegulationCPSF30 and TTP are non-classical zinc finger proteins (ZFS) that contain domains with a CCCH motif. CPSF30 has 5 CCCH domains and TTP has 2 CCCH domains. Both proteins are involved in RNA regulation; CPSF30 regulates pre-mRNA and TTP regulates mRNA; however, only TTP has been shown to directly bind to RNA (via its CCCH domain, targeting AU-rich sites). Given the sequence similarity between TTP and CPSF30, we hypothesized that CPSF30 directly binds AU-rich RNA sequences via its CCCH domains. To test this hypothesis, a construct of CPSF30 containing the five CCCH domains, was over-expressed and purified. Unexpectedly, CPSF30 was reddish in color, suggesting iron coordination. UV-visible, ICP-MS analysis and XAS spectroscopy revealed that the protein contains a 2Fe-2S cluster in addition to four zinc domains. The 2Fe-2S cluster utilizes a CCCH ligand set, and is the second example of this site in biology! RNA binding studies, using EMSA and fluorescence anisotropy (FA), with ?-synuclein AU-rich pre-mRNA as a target, were then performed. From these studies, we determined that (1) CPSF30 binds directly to AU-rich targets on pre-mRNA via a cooperative binding mechanism and (2) CPSF30 requires both iron and zinc coordination for RNA binding. Studies focused on Cu(I) binding to TTP will also be presented. Cu(I) is toxic in excess and there is emerging evidence that ZF sites may be target of Cu(I) toxicity. Using UV-visible and circular dichroism spectroscopies, we have determined that 3 Cu(I) ions bind to TTP and that Cu(I) binding inhibits the structure of the protein. In addition, RNA binding studies, using FA with the TNF-? AU-rich mRNA revealed that Cu(I) inhibits the TTP-RNA interaction. We propose that inhibition of TTP function by Cu(I) contributes to its mechanism of toxicity.