Pritts, Jordan Dylan2022-06-142022-06-142022http://hdl.handle.net/10713/19171University of Maryland, Baltimore. Pharmaceutical Sciences. Ph.D. 2022.Zinc Finger (ZF) proteins utilize zinc as a structural co-factor. ZFs are classed based upon the amino acid ligands that coordinate Zn. One class of ZFs is the CCCH class, which uses three cysteine and one histidine as zinc coordinating ligands. These proteins regulate RNA via a ZF/RNA binding interaction. One member of the CCCH class of ZFs is cleavage and polyadenylation specificity factor 30 (CPSF30) which contains 5 CCCH domains. In addition, CPSF30 has a ‘CCHC’ or zinc knuckle domain. CPSF30 regulates pre-mRNA processing. Experiments to determine the RNA recognition properties of CPSF30 and the role of an unusual Fe-S co-factor have been performed. A construct of CPSF30 that contains the 5 CCCH domains binds to an RNA sequence – AAUAAA – which is also called the polyadenylation signal (PAS) and present in a majority of pre-mRNA. I determined that mutations to the PAS, including some associated with human diseases, result in binding affinity changes to CPSF30 suggesting a connection between RNA binding and disease states. I isolated and characterized full length CPSF30, which contains the CCHC domain along with the 5 CCCH domains, for the first time. A major finding was that the CCHC zinc knuckle domain binds polyU RNA. Thus, CPSF30 appears to have bipartite RNA recognition. I determined that RNA recognition to these two distinct RNA sequences by CPSF30 is a competitive event and proposed a model of CPSF30/RNA binding related to alternative polyadenylation or cytoplasmic polyadenylation. The Fe-S cluster of CPSF30 was characterized. Mössbaauer and XAS spectroscopy data support a 2Fe-2S cluster with a CCCH ligand set. Reduction with dithionite followed by UV-visible and EPR spectroscopies demonstrated that the protein is redox active. Metal coupled protein oxidation/mass spectrometry indicate that ZF2 of CPSF30 is the site of the Fe-S co-factor.en-USCSPF302Fe-2S ClusterZinc FingersLigandsBase Sequencedissertation2022-06-10Michel, Sarah L. J.