title: Cleavage and polyadenylation specificity factor 30: An RNA-binding zinc-finger protein with an unexpected 2Fe-2S cluster.
abstract: Cleavage and polyadenylation specificity factor 30 (CPSF30) is a key protein involved in pre-mRNA processing. CPSF30 contains five Cys3His domains (annotated as "zinc-finger" domains). Using inductively coupled plasma mass spectrometry, X-ray absorption spectroscopy, and UV-visible spectroscopy, we report that CPSF30 is isolated with iron, in addition to zinc. Iron is present in CPSF30 as a 2Fe-2S cluster and uses one of the Cys3His domains; 2Fe-2S clusters with a Cys3His ligand set are rare and notably have also been identified in MitoNEET, a protein that was also annotated as a zinc finger. These findings support a role for iron in some zinc-finger proteins. Using electrophoretic mobility shift assays and fluorescence anisotropy, we report that CPSF30 selectively recognizes the AU-rich hexamer (AAUAAA) sequence present in pre-mRNA, providing the first molecular-based evidence to our knowledge for CPSF30/RNA binding. Removal of zinc, or both zinc and iron, abrogates binding, whereas removal of just iron significantly lessens binding. From these data we propose a model for RNA recognition that involves a metal-dependent cooperative binding mechanism.
authors.affiliation: Department of Pharmaceutical Sciences, School of Pharmacy, University of Maryland, Baltimore, MD 21201;
authors.forename: Geoffrey D
authors.initials: GD
authors.lastname: Shimberg
authors.affiliation: Department of Pharmaceutical Sciences, School of Pharmacy, University of Maryland, Baltimore, MD 21201;
authors.forename: Jamie L
authors.initials: JL
authors.lastname: Michalek
authors.affiliation: Department of Pharmaceutical Sciences, School of Pharmacy, University of Maryland, Baltimore, MD 21201;
authors.forename: Abdulafeez A
authors.initials: AA
authors.lastname: Oluyadi
authors.affiliation: Department of Pharmaceutical Sciences, Wayne State University, Detroit, MI 48201;
authors.forename: Andria V
authors.initials: AV
authors.lastname: Rodrigues
authors.affiliation: Department of Biochemistry and Molecular Biology, School of Medicine, University of Maryland, Baltimore, MD 21201.
authors.forename: Beth E
authors.initials: BE
authors.lastname: Zucconi
authors.affiliation: Department of Pharmaceutical Sciences, School of Pharmacy, University of Maryland, Baltimore, MD 21201;
authors.forename: Heather M
authors.initials: HM
authors.lastname: Neu
authors.affiliation: Department of Pharmaceutical Sciences, School of Pharmacy, University of Maryland, Baltimore, MD 21201;
authors.forename: Shanchari
authors.initials: S
authors.lastname: Ghosh
authors.affiliation: Department of Pharmaceutical Sciences, School of Pharmacy, University of Maryland, Baltimore, MD 21201;
authors.forename: Kanisha
authors.initials: K
authors.lastname: Sureschandra
authors.affiliation: Department of Biochemistry and Molecular Biology, School of Medicine, University of Maryland, Baltimore, MD 21201.
authors.forename: Gerald M
authors.initials: GM
authors.lastname: Wilson
authors.affiliation: Department of Pharmaceutical Sciences, Wayne State University, Detroit, MI 48201;
authors.forename: Timothy L
authors.initials: TL
authors.lastname: Stemmler
authors.affiliation: Department of Pharmaceutical Sciences, School of Pharmacy, University of Maryland, Baltimore, MD 21201; smichel@rx.umaryland.edu.
authors.forename: Sarah L J
authors.initials: SL
authors.lastname: Michel
mesh_terms: D001665:Binding Sites; D039223:Cleavage And Polyadenylation Specificity Factor / Q000737:chemistry* / Q000235:genetics; D003198:Computer Simulation; D006801:Humans; D007501:Iron / Q000737:chemistry*; D008956:Models, Chemical; D026723:Polyadenylation / Q000235:genetics; D011485:Protein Binding; D039104:RNA 3' Polyadenylation Signals / Q000235:genetics*; D012333:RNA, Messenger / Q000737:chemistry*; D013455:Sulfur / Q000737:chemistry*; D039221:mRNA Cleavage and Polyadenylation Factors / Q000737:chemistry* / Q000235:genetics
chemical_list: C000594014:CPSF4 protein, human; D039223:Cleavage And Polyadenylation Specificity Factor; D012333:RNA, Messenger; D039221:mRNA Cleavage and Polyadenylation Factors; D013455:Sulfur; D007501:Iron
keywords: RNA; binding; iron–sulfur; protein; zinc