Functional Role of Zinc in Poly(A) Synthesis Catalyzed by Nuclear Poly(A) Polymerase

Rabbits were immunized with either RNA polymerase I or poly(A) polymerase that had been purified to apparent homogeneity and was devoid of nucleic acids. Sera from rabbits thus immunized were screened for antibodies against nucleic acids. All seven rabbits injected with RNA polymerase I but none of the four rabbits immunized with poly(A) polymerase produced anti-nucleic acid antibodies. Anti-RNA polymerase I antibodies were induced after a single injection of the enzyme. Anti-polynucleotide antibodies were not detectable until after the second immunization. Anti-RNA polymerase I antibodies could be detected with as little as 100 pg of purified RNA polymerase I in the radioimmunoassay. At least 50 ng of poly(A) or 200 ng of DNA was required to detect anti-nucleic acid antibodies. The immunoreactivity of anti-RNA polymerase I antisera was greater with synthetic polynucleotides than with DNA, particularly early in the immunization schedule. Alkaline phosphatase treatment of poly(A) to remove 5' phosphates nearly abolished its antigenicity with respect to the early sera and decreased antibody binding of later sera by 60%. These results indicate that the anti-nucleic acid antibodies produced early were primarily directed against determinants including the 5'-terminal phosphates while antibodies produced later were directed against other sites. The antinucleic acid antibodies and anti-RNA polymerase I antibodies formed two distinct populations that were not immunologically crossreactive. We suggest that after injection, RNA polymerase I becomes associated with the nucleic acids present in blood plasma which renders them immunogenic; thus, association of nucleic acids with autoimmunogenic RNA polymerase I may be one of the mechanisms by which anti-DNA antibodies are induced in systemic lupus erythematosus.Sera from patients with systemic lupus erythematosus (SLE) and other rheumatic autoimmune diseases contain antibodies against a variety of proteins and nucleic acids in the cell nucleus (reviewed in ref. 1). It has been postulated that phosphodiester bonds of nucleic acids are autoantigenic determinants in both human SLE (2) and the murine lupuslike syndrome (3). Recently, we identified RNA polymerase I (4) and protein kinase NII (5) as target nuclear antigens in SLE, rheumatoid arthritis, and mixed connective tissue disease and demonstrated (6) that phosphorylation of the polymerase can dramatically increase its antigenicity with respect to SLE patient autoantibodies. The importance of phosphate groups in the autoantigenicity of both RNA polymerase I and DNA and the recent reports that anti-DNA autoantibodies were capable of reacting with phospholipids (2, 3) and proteins (7) suggested that anti-RNA polymerase I antibodies might crossreact with DNA. In addition, since RNA polymerase I can interact with DNA as part of its biological function, it is plausible that association of the immunogenic enzyme with DNA, which is present in blood plasma (8), might induce production of anti-DNA antibodies. These possibili...

Section: Methodssupporting

confidence: 86%

Immunization of rabbits with purified RNA polymerase I induces a distinct population of antibodies against nucleic acids as well as anti-RNA polymerase I antibodies, both characteristic of systemic lupus erythematosus.

Stetler¹,

Jacob²

1985

“…Wells prepared in the absence of enzyme served as controls. In some instances, rat hepatoma poly(A) polymerase, purified as described (19,20), was adsorbed to the wells in place of RNA polymerase I as an additional control. Antisera produced by rabbits immunized with RNA polymerase I (17) efficiently bound to the immobiAbbreviations: SLE, systemic lupus erythematosus; ANA, antinuclear antibody(ies); ENA, extractable nuclear antigens; MCTD, mixed connective tissue disease; RA, rheumatoid arthritis; RNP, ribonucleoprotein; RIA, radioimmunoassay; DBM, diazobenzyloxymethyl.…”

Section: Methodsmentioning

confidence: 99%

Antibodies to distinct polypeptides of RNA polymerase I in sera from patients with rheumatic autoimmune disease.

Stetler¹,

Rose²,

Wenger³

et al. 1982

Sera from patients with rheumatic autoimmune diseases were screened for antibodies directed against RNA polymerase I by using a solid-phase radioimmunoassay. Significant levels of the antibodies were detected in the sera of all patients with either systemic lupus erythematosus or mixed connective tissue disease and in 78% of the individuals with rheumatoid arthritis. No detectable anti-RNA polymerase I antibodies were found in the sera from healthy subjects. Individuals taldng hydralazine, three of whom exhibited symptoms of drug-induced lupus, had barely detectable levels of the antibodies. Immunoglobulins obtained from sera containing anti-RNA polymerase I antibodies, as determined by the radioimmunoassay, could inhibit RNA polymerase

“…Poly(A) polymerase was purified from isolated nuclei of Morris hepatoma 3924A (a solid rat tumor) by successive chromatography on DEAE-Sephadex, phosphocellulose, hydroxyapatite, and QAE-Sephadex (8). After QAE-Sephadex chromatography, the enzyme was purified to homogeneity on DNA-cellulose (9). Poly(A) polymerase assays were performed as indicated in ref.…”

Section: Methodsmentioning

confidence: 99%

“…Recent studies have suggested a role for poly(A) tracts in the stabilization ofmRNA (4), in gene splicing (5,6), and in the formation of the termination codon for translation (7). Studies in our laboratory have shown that nuclear poly(A) polymerase from rat liver and a rat hepatoma differ with respect to the apparent Km for ATP, primer saturation levels, sensitivity to pH changes, molecular weight, amino acid composition (8), zinc content (9), and degree ofphosphorylation (10). Moreover, recent immunological data have demonstrated that hepatoma poly(A) polymerase is much more closely related to the enzyme from other tumors and fetal liver than to the normal liver protein (11).…”

mentioning

confidence: 99%

Anti-poly(A) polymerase antibodies in sera of tumor-bearing rats and human cancer patients.

Stetler¹,

Rose²,

Jacob³

1981

Poly(A) polymerase (polynucleotide adenylyltransferase; ATP:polynucleotide adenylyltransferase, EC 2.7.7.19) was covalently linked to diazobenzyloxymethyl-filters and used to screen the sera from a number of tumor-bearing rats and human cancer patients for antibodies to poly(A) polymerase. Sera from rats that had been inoculated with any of several Morris hepatomas or a mammary adenocarcinoma contained immunoglobulins capable of complexing with poly(A) polymerase. No antibodies to the enzyme could be detected in sera from control animals or from those bearing tumors for short periods of time. Antibodies to poly(A) polymerase were also observed in sera from human patients with leukemia, polycythemia vera, and Wilms tumor. The antibodies were not evident in sera from normal volunteers or from patients with nonneoplastic diseases. These included lupus erythematosus, a disorder in which antibodies are produced against an array of nuclear proteins. Immunoglobulins from the serum of one of the human patients were capable of inhibiting poly(A) polymerase activity in vitro, whereas those prepared from the serum of a normal volunteer did not affect enzyme activity. As determined by the diazobenzyloxymethyl-filter technique, the relative concentration of antibodies in the sera of an individual with leukemia (in remission) increased severalfold during a relapse. These data suggest that the presence ofantibodies to poly(A) polymerase may be characteristic of sera from cancer patients and that the relative concentration of these antibodies may be indicative of the disease state.Among the several steps involved in the processing of eukaryotic mRNA is the addition of poly(A) tracts to the 3'-OH termini (1). Poly(A) polymerase (polynucleotide adenylyltransferase; ATP:polynucleotide adenylyltransferase, EC 2.7.7.19), the enzyme that catalyzes the polyadenylylation reaction, has been isolated from a variety of mammalian sources and cell fractions (for reviews, see refs. 2 and 3). Recent studies have suggested a role for poly(A) tracts in the stabilization ofmRNA (4), in gene splicing (5,6), and in the formation of the termination codon for translation (7). Studies in our laboratory have shown that nuclear poly(A) polymerase from rat liver and a rat hepatoma differ with respect to the apparent Km for ATP, primer saturation levels, sensitivity to pH changes, molecular weight, amino acid composition (8), zinc content (9), and degree ofphosphorylation (10). Moreover, recent immunological data have demonstrated that hepatoma poly(A) polymerase is much more closely related to the enzyme from other tumors and fetal liver than to the normal liver protein (11). These observations raised the possibilities that autochthonous tumor poly(A) polymerase may be immunogenic and that sera from donors with neoplastic disease may contain anti-poly(A) polymerase antibodies. We now report that such antibodies are present in the sera from tumor-bearing animals as well as from human cancer patients. METHODSPurification of Poly(A) Polymerase. Po...