An examination of the metal ion content in the active sites of human endonucleases CPSF73 and INTS11

Huang, Ji; Liu, Xiangyang; Sun, Yadong; Li, Zhuang; Lin, Min-Han; Hamilton, Keith; Mandel, Corey R.; Sandmeir, Felix; Conti, Elena; Oyala, Paul H.; Tong, Liang

doi:10.1016/j.jbc.2023.103047

An examination of the metal ion content in the active sites of human endonucleases CPSF73 and INTS11

Journal Article · Wed Feb 22 00:00:00 EST 2023 · Journal of Biological Chemistry

DOI:https://doi.org/10.1016/j.jbc.2023.103047· OSTI ID:2423281

^[1]; Liu, Xiangyang ^[1]; ^[1]; Li, Zhuang ^[1]; ^[1]; Hamilton, Keith ^[1]; Mandel, Corey R. ^[1]; ^[2]; Conti, Elena ^[2]; ^[3]; ^[1]

Columbia Univ., New York, NY (United States)
Max Planck Institute of Biochemistry, Martinsried (Germany)
California Institute of Technology (CalTech), Pasadena, CA (United States)

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-β-lactamase domain and a β-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 processing 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 endonuclease 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.

View Accepted Manuscript (DOE)

Research Organization:: Argonne National Laboratory (ANL), Argonne, IL (United States). Advanced Photon Source (APS)

Sponsoring Organization:: National Institute of General Medical Sciences (NIGMS); USDOE

Grant/Contract Number:: AC02-06CH11357

OSTI ID:: 2423281

Journal Information:: Journal of Biological Chemistry, Journal Name: Journal of Biological Chemistry Journal Issue: 4 Vol. 299; ISSN 0021-9258

Publisher:: American Society for Biochemistry and Molecular BiologyCopyright Statement

Country of Publication:: United States

Language:: English

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37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CHEMISTRY
59 BASIC BIOLOGICAL SCIENCES
Biochemistry & Molecular Biology
CPSF73
Endonuclease
ICP-MS
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Integrator
Metal ions
RNA processing

An examination of the metal ion content in the active sites of human endonucleases CPSF73 and INTS11

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References (53)

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