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Free keywords:
PRE-MESSENGER-RNA; POLYADENYLATION FACTOR CPSF-73; BETA-LACTAMASE;
PIGGYBAC TRANSPOSASE; STRUCTURAL INSIGHTS; INTEGRATOR COMPLEX; 3'-END;
END; RECONSTITUTION; MECHANISMBiochemistry & Molecular Biology;
Abstract:
Human cleavage and polyadenylation specificity factor (CPSF)73 (also known as CPSF3) is the endoribonuclease that catalyzes the cleavage reaction for the 3'-end processing of pre-mRNAs. The active site of CPSF73 is located at the interface between a metallo-beta-lactamase domain and a beta-CASP domain. Two metal ions are coordinated by conserved residues, five His and two Asp, in the active site, and they are critical for the nuclease reaction. The metal ions have long been thought to be zinc ions, but their exact identity has not been examined. Here we present evidence from inductively coupled plasma mass spectrometry and X-ray diffraction analyses that a mixture of metal ions, including Fe, Zn, and Mn, is present in the active site of CPSF73. The abundance of the various metal ions is different in samples prepared from different expression hosts. Zinc is present at less than 20% abundance in a sample expressed in insect cells, but the sample is active in cleaving a pre-mRNA substrate in a reconstituted canonical 3'-end pro-cessing machinery. Zinc is present at 75% abundance in a sample expressed in human cells, which has comparable endonuclease activity. We also observe a mixture of metal ions in the active site of the CPSF73 homolog INTS11, the endo-nuclease for Integrator. Taken together, our results provide further insights into the role of metal ions in the activity of CPSF73 and INTS11 for RNA 3'-end processing.
