• Cys3His Zinc Finger Proteins: Metal Ion Coordination and RNA Recognition Properties of Tristetraprolin and the 30 kDa Subunit of the Cleavage and Polyadenylation Specificity Factor

      Michalek, Jamie Leigh; Michel, Sarah L. J. (2013)
      Zinc finger proteins (ZFs) utilize zinc ions to fold and function. ZFs globally regulate gene expression through interactions with DNA, RNA and other proteins. The Cys₃His ZFs are an emerging class of ZFs that regulate RNA. Tristetraprolin (TTP) and the 30 kDa subunit of the cleavage and polyadenylation specificity factor (CPSF30) are two examples of Cys₃His ZFs. TTP binds to adenosine/uridine-rich sequences found in the 3'-untranslated regions of cytokine mRNA. TTP is a potential target for cadmium toxicity. A construct of TTP (TTP-2D) was over-expressed and purified, and the Cd(II) binding properties were determined using UV-visible spectroscopy. TTP-2D was found to bind Cd(II) with a Kd of 3.5 (± 0.1) x 10⁻⁹ M at a 2:1 Cd(II):TTP-2D stoichiometry. Cd(II)-TTP-2D selectively bound to an AU-rich RNA sequence, and exhibited greater selectivity than its Zn(II), Fe(II) or Fe(III)-TTP-2D counterparts, as measured by Fluorescence Anisotropy (FA). CPSF30 is essential for pre-mRNA processing and is predicted to interact with the pre-mRNA polyadenylation signal, AAUAAA. Constructs of CPSF30 that contain the whole protein, its 5 Cys₃His domains and its singular Cys₂HisCys domain were overexpressed and purified. These constructs coordinate Co(II) and Zn(II) at the expected 6:1, 5:1 and 1:1 M(II):protein stoichiometry with affinities ( K<sub>d</sub>s) of 6.3 (± 0.6) x 10⁵ M, 5.1 (± 0.5) x 10⁵ M and 3.1 (± 0.1) x 10⁵ M for Co(II). To increase solubility, a maltose binding fusion (MBP) of the 5 Cys₃His domain, called MBP-CPSF30-5FE, was overexpressed and purified in the folded state. MBP-CPSF30-5FE was found to contain with 4 zinc and 1 iron ions by Inductively Coupled Plasma Mass Spectrometry (ICP-MS). MBP-CPSF30-5FE was determined to selectively bind to AU-rich RNA target from α-Synuclein pre-mRNA, using a combination of electrophoretic mobility shift assays (EMSA) and FA. The binding data was best fit to a cooperative binding model with 2 protein:1 RNA with Kds of 1.44 (± 0.04) x 10⁷ M for α-Synuclein24, 1.15 (± 0.04) x 10⁷ M for α-Synuclein30 and 9.35 (± 0.27) x 10⁸M for α-Synuclein38 and an average hill coefficient of 1.63 (± 0.07).