Identification of the Site of the Fe–S Cluster in CPSF30 via Metal-Coupled Oxidation-Mass Spectrometry

Abstract

Cleavage and polyadenylation specificity factor 30 (CPSF30) is a zinc finger protein that plays a key role in pre-mRNA processing by recognizing specific AU-rich sequence elements to facilitate polyadenylation. CPSF30 contains five highly conserved domains made up of three cysteines and a single histidine residue (CCCH, where C = cysteine; H = histidine). These domains coordinate both Zn and an essential 2Fe-2S cluster required for high-affinity RNA binding. The location of the 2Fe-2S cluster within the five CCCH domains and its function have proven elusive. Here, we apply metal-catalyzed oxidation mass spectrometry (MCO-MS) to identify the 2Fe-2S cluster site within CPSF30. By first determining its redox properties and then initiating Fenton chemistry, we localized the Fe–S cluster to the second CCCH domain. The effect of redox state on RNA binding was then assessed, and it was determined that the Fe–S site is a structural domain. The effect of redox status in THP-1 cells on CPSF30 was then investigated, and the redox properties of the cluster were linked to protein abundance under hypoxic and normoxic stress. Together, this work describes a new approach to identify Fe–S sites in complex metalloproteins that house multiple metal cofactors, suggests a mechanism for metal-mediated RNA recognition by CPSF30, and reveals redox-mediated control of CPSF30 in cells.