Determination of the Mechanism of RNA Regulation by CPSF30 Utilizing Both Biophysical and Structural Approaches

Zinc finger proteins are metal regulated transcription factors that utilize zinc to fold and function. ZFs provide high specificity DNA, RNA, and protein binding resulting in gene regulation at both the transcriptional and translational levels. ZF proteins utilize 4 cysteine/histidine ligands to bind Zn ions and can consist of 1 or more domains. These proteins are commonly classed based on the ratios of cysteine to histidine ligands as well as their spacing in the amino acid sequence. We are investigating Cleavage and Polyadenylation Specificity Factor 30 (CPSF30). Mammalian CPSF30 contains 5 CCCH type ZF domains and 1 ‘zinc knuckle’ domain and is vital to proper polyadenylation. We have recently shown that CPSF30 is an RNA binding protein involved in recognition of the AAUAAA polyadenylation signal (PAS) found in the majority of eukaryotic pre-mRNAs. Interestingly, we found CPSF30 to harbor a 2Fe-2S cluster as well as traditional zinc binding sites. This is one of a few emerging examples of improper ZF annotations following the whole genome sequencing efforts at the turn of the century. Current work aims to characterize the RNA binding recognition mechanism of CPSF30 as well as the role and redox properties of the incorporated 2Fe-2S cluster. Fluorescence anisotropy is being utilized to look at RNA mutational effects on binding and recognition while hydrogen/deuterium exchange coupled mass spectrometry is being employed to determine which ZF domains of CPSF30 are involved in binding. Additionally, these studies are tied together with ongoing crystallography efforts for the interaction.