[CANCER RESEARCH 49, 2827-2833, June 1, 1989] Review Polyadenylate Polymerases from Normal and Cancer Cells and Their Potential Role in Messenger RNA Processing: A Review1 Samson T. Jacob,2 Michael P. Terns,2 and Kathleen A. Maguire3 Department of Pharmacology and Cell and Molecular Biology Center, The Pennsylvania State University, College of Medicine, Hershey, Pennsylvania 17033 Abstract Two structurally and immunologically distinct species of nuclear poh - adenylate (poly(A)) polymerases have been characterized. One of these enzymes is relatively absent in normal tissues but is predominant in primary and transplanted tumors and transformed cell lines. The presence of the tumor type enzyme in fetal liver, but not in regenerating liver, suggests that it is an oncofetal protein. Antibodies against the tumor- type poly(A) polymerases are present in the sera of rats bearing tumors and in some cancer patients. These antibodies are also found in the sera of rats fed hepatocarcinogen even before preneoplastic nodules were visible, which suggests that elicitation of these antibodies is an early event in neoplastic transformation. Autoantibodies against both liver- type and tumor-type poly(A) polymerase are also present in some rheu matic autoimmune sera. Polyclonal antibodies against purified enzyme from a rat hepatoma, which exhibit a single band upon immunoblot analysis, were used in cell-free extracts to study the role of poly(A) polymerase in the 3'-end processing of pre-mRNA. These studies showed that the antibodies blocked both endonucleolytic cleavage and poly(A) addition at the cleavage site and complex formation between factors in the extract and pre-mRNA. Independent studies in other laboratories have demonstrated that both the cleavage and poly(A) polymerase activ ities require the same component for their function. These observations suggest that both cleavage and polyadenylation reactions are tightly coupled in a functional complex. I. Introduction The vast majority of eukaryotic mRNAs contain poly(A)4 tails at their 3' ends. Despite the significant progress made towards the identification and partial characterization of a few factors involved in the cleavage/polyadenylation of pre- mRNAs, the mechanism by which a properly polyadenylated mRNA 3' terminus is formed has not been fully elucidated. Current evidence indicates that RNA polymerase H-directed transcription proceeds well beyond the poly(A) site and termi nates over a heterogeneous DNA stretch at considerable dis tances from the eventual 3' end. The 3' terminus of mRNA arises from a posttranscriptional reaction which involves an endonucleolytic cleavage of the larger precursor followed by the addition of an adenosine tract, approximately 200-250 nucle- otides long, to this newly generated end (for extensive reviews, see refs. 1-3). It is presumed that the latter reaction is enzy- matically accomplished by poly(A) polymerase (polynucleotide adenyltransferase; ATP: polynucleotide adenyltransferase, EC 2.7.7.19). This review focuses on the important characteristics of poly(A) polymerase and the recent studies which have pro- Received10/28/88;revised2/21/89;accepted2/27/89. Thecostsof publicationof thisarticleweredefrayedin partbythepayment ofpagecharges.Thisarticlemustthereforebeherebymarkedadvertisement in accordancewith18U.S.C.Section1734solelytoindicatethisfact. 1Work carried out in the authors' laboratory was supported by USPHS Grants CA 25078 and CA 31894, by the Pennsylvania Lupus Foundation, and by the Diagnostic Division of Allied Health and Scientific Products (to S. T. J.). 2 Present address: Department of Pharmacology and Molecular Biology, The Chicago Medical School, N. Chicago, IL 60064. 3 Present address: The Wistar Institute, Spruce St., Philadelphia, PA. 4 The abbreviations used are: poly(A), polyadenylate; RNP, ribonucleoprotein; sn, small nuclear. vided direct evidence for the role of this well characterized enzyme in the 3' end processing of eukaryotic pre-mRNAs. II. Summary of the Properties of Poly(A) Polymerase For extensive discussions on the characteristics of poly(A) polymerase the reader is asked to refer to previous reviews (see Refs. 4 and 5). These reviews have covered in great detail intracellular distribution, size and subunits, other physical properties, substrate, primer and divalent metal ion require ments, inhibitors, phosphorylation of enzyme, and nucleases associated with enzyme preparations. Only a brief description of the properties of this enzyme from higher organisms that are relevant to the present discussion is presented here. Poly(A) polymerase appears to be a ubiquitous enzyme and is present in all organisms ranging from prokaryotes to eukar- yotes. In mammalian cells, the enzyme has been identified in the nucleus (6), RNPs (7), mitochondria (8), microsomes (9), ribosomes (10), and postmitochondrial fractions (11). The nu clear enzyme is found exclusively in the extranucleolar fraction, which is consistent with the lack of polyadenylation of rRNA in the nucleolus. The nuclear enzyme occurs as chromatin- bound and free forms (12-16). Following solubilization of the bound enzyme, it attains the characteristics of the "free" en zyme, which indicates that the two populations of the enzyme represent the same polypeptide in two functional states. The chromatin-bound enzyme is highly sensitive to low levels of cordycepin triphosphate (3'-dATP) whereas the free form of the enzyme is inhibited only by relatively high concentrations of the ATP analogue (17). The differential sensitivity of the two poly(A) polymerase populations to 3'-dATP is consistent with similar responses of the initial polyadenylation and poly(A) extension reactions to cordycepin in vivo (18, 19). Based on these data, we have concluded that the chromatin-bound and free forms of the enzyme are responsible for the initial polyad enylation and poly(A) elongation reactions, respectively (17, 20). More recent studies with a reconstitution system in vitro have further supported this contention (see Section VIII). Purification of enzymes consisting of a single subunit has been accomplished in several animal systems (for review, see Refs. 4, 5, 21, and 22). Poly(A) polymerase is assayed usually by monitoring the incorporation of labeled AMP into synthetic primer. It is clear that purified poly(A) polymerase cannot catalyze the entire reaction which leads to mRNA 3' end formation. Poly(A) polymerase activity is only one of the activ ities required for this highly specific and complex reaction. The size of the enzyme, as displayed after gel electrophoresis under denaturing conditions, ranges from 37,000 to 60,000 depending upon the source of the enzyme (see Refs. 4 and 5). The enzyme can occur as a dimer or tetramer (see Section III). Very high molecular weights (300,000) have been reported under certain conditions (see Section IV). The enzyme has displayed heterogeneity upon chromatography on phosphocel- lulose (23, 24) or carboxymethyl cellulose (25) columns. Al- 2827 on July 29, 2015. © 1989 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from