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Molecular and Cellular Biology, August 2001, p. 5614-5623, Vol. 21, No. 16
0270-7306/01/$04.00+0   DOI: 10.1128/MCB.21.16.5614-5623.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.

Identification and Functional Characterization of Neo-Poly(A) Polymerase, an RNA Processing Enzyme Overexpressed in Human Tumors

Suzanne L. Topalian,1,* Syuzo Kaneko,2 Monica I. Gonzales,1 Gareth L. Bond,2 Yvona Ward,3 and James L. Manley2

Surgery Branch1 and Medicine Branch,3 National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892, and Department of Biological Sciences, Columbia University, New York, New York 100272

Received 17 April 2001/Returned for modification 16 May 2001/Accepted 29 May 2001

Poly(A) polymerase (PAP) plays an essential role in polyadenylation of mRNA precursors, and it has long been thought that mammalian cells contain only a single PAP gene. We describe here the unexpected existence of a human PAP, which we call neo-PAP, encoded by a previously uncharacterized gene. cDNA was isolated from a tumor-derived cDNA library encoding an 82.8-kDa protein bearing 71% overall similarity to human PAP. Strikingly, the organization of the two PAP genes is nearly identical, indicating that they arose from a common ancestor. Neo-PAP and PAP were indistinguishable in in vitro assays of both specific and nonspecific polyadenylation and also endonucleolytic cleavage. Neo-PAP produced by transfection was exclusively nuclear, as demonstrated by immunofluorescence microscopy. However, notable sequence divergence between the C-terminal domains of neo-PAP and PAP suggested that the two enzymes might be differentially regulated. While PAP is phosphorylated throughout the cell cycle and hyperphosphorylated during M phase, neo-PAP did not show evidence of phosphorylation on Western blot analysis, which was unexpected in the context of a conserved cyclin recognition motif and multiple potential cyclin-dependent kinase (cdk) phosphorylation sites. Intriguingly, Northern blot analysis demonstrated that each PAP displayed distinct mRNA splice variants, and both PAP mRNAs were significantly overexpressed in human cancer cells compared to expression in normal or virally transformed cells. Neo-PAP may therefore be an important RNA processing enzyme that is regulated by a mechanism distinct from that utilized by PAP.

* Corresponding author. Mailing address: Surgery Branch, National Cancer Institute, NIH 10/2B47, Bethesda, MD 20892. Phone: (301) 496-4269. Fax: (301) 402-0922. E-mail: Suzanne_Topalian{at}nih.gov.

Molecular and Cellular Biology, August 2001, p. 5614-5623, Vol. 21, No. 16
0270-7306/01/$04.00+0   DOI: 10.1128/MCB.21.16.5614-5623.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.


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