CPSF30, a Novel Non-Classical Zinc Finger Protein that Utilizes Iron & Zinc Coordination for RNA Recognition

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Zinc finger (ZF) proteins are eukaryotic proteins that are involved in transcriptional and translational regulation. ZFs utilize cysteine and histidine residues to coordinate zinc ions to fold and function properly. There are fourteen different classes of zinc finger proteins, which are classified based upon the ligands involved in zinc coordination, the ZF domain folds, and the proteins’ biological targets. Cleavage and Polyadenylation Specificity Factor 30 (CPSF30) is a ‘non-classical’ ZF protein that contains multiple Cys3His zinc binding domains and is involved in pre-mRNA regulation. CPSF30 contains 5 Cys3His domains and a Cys2HisCys zinc knuckle domain. A construct of CPSF30 construct containing only CPSF30's five Cys3His domains, denoted as CPSF305FE was prepared. This protein is predicted to have five zinc ions; however, UV-visible and ICP-MS analysis of the isolated construct revealed that CPSF305FE contains iron in addition to zinc in a 1:4:1 Fe:Zn:CPSF305FE stoichiometetry. XAS analysis identified the iron site as a 2Fe-2S cluster. CPSF30 is predicted to recognize AU-rich RNA sequences. To test this hypothesis, RNA binding studies using both electrophoretic mobility shift assays (EMSA) and fluorescence anisotropy (FA) of CPSF305FE with α-synuclein pre-mRNA (which contains an AU-rich sequence) along with a series of mutated RNA sequences were performed. These studies revealed that CPSF30 binds to α-synuclein pre-RNA with nanomolar affinity and requires the AU-rich sequence for binding. The binding interaction was best fit to a cooperative binding model, suggesting that the CPF30/RNA binding event is cooperative.