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

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.