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Molecular and Cellular Biology, January 2009, p. 31-42, Vol. 29, No. 1
0270-7306/09/$08.00+0     doi:10.1128/MCB.00776-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Studies of the 5' Exonuclease and Endonuclease Activities of CPSF-73 in Histone Pre-mRNA Processing ^,

Xiao-cui Yang, Kelly D. Sullivan, William F. Marzluff, and Zbigniew Dominski^*

Department of Biochemistry and Biophysics and Program in Molecular Biology and Biotechnology, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599

Received 14 May 2008/ Returned for modification 9 July 2008/ Accepted 15 October 2008

Processing of histone pre-mRNA requires a single 3' endonucleolytic cleavage guided by the U7 snRNP that binds downstream of the cleavage site. Following cleavage, the downstream cleavage product (DCP) is rapidly degraded in vitro by a nuclease that also depends on the U7 snRNP. Our previous studies demonstrated that the endonucleolytic cleavage is catalyzed by the cleavage/polyadenylation factor CPSF-73. Here, by using RNA substrates with different nucleotide modifications, we characterize the activity that degrades the DCP. We show that the degradation is blocked by a 2'-O-methyl nucleotide and occurs in the 5'-to-3' direction. The U7-dependent 5' exonuclease activity is processive and continues degrading the DCP substrate even after complete removal of the U7-binding site. Thus, U7 snRNP is required only to initiate the degradation. UV cross-linking studies demonstrate that the DCP and its 5'-truncated version specifically interact with CPSF-73, strongly suggesting that in vitro, the same protein is responsible for the endonucleolytic cleavage of histone pre-mRNA and the subsequent degradation of the DCP. By using various RNA substrates, we define important space requirements upstream and downstream of the cleavage site that dictate whether CPSF-73 functions as an endonuclease or a 5' exonuclease. RNA interference experiments with HeLa cells indicate that degradation of the DCP does not depend on the Xrn2 5' exonuclease, suggesting that CPSF-73 degrades the DCP both in vitro and in vivo.

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* Corresponding author. Mailing address: Program in Molecular Biology and Biotechnology, CB #3280, University of North Carolina, Chapel Hill, NC 27599. Phone: (919) 843-0307. Fax: (919) 962-1274. E-mail: dominski{at}med.unc.edu

Published ahead of print on 27 October 2008.

Supplemental material for this article may be found at http://mcb.asm.org/.

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Molecular and Cellular Biology, January 2009, p. 31-42, Vol. 29, No. 1
0270-7306/09/$08.00+0     doi:10.1128/MCB.00776-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

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