Cell-free synthesis of tumor-type poly(A) polymerase

Previous studies in this laboratory suggested that in adult liver, either the gene for the tumor-type poly(A) polymerase is poorly transcribed or the mRNA for this enzyme is largely not expressed. To test these possibilities, total RNA from rat liver and Morris hepatoma 3924A RNA were isolated by using a guanidine thiocyanate method; poly(A+) RNA and poly(A-) RNA were separated by oligo(dT)-cellulose chromatography and used for translation in a rabbit reticulocyte lysate system. After in vitro translation, the products were immunoprecipitated with either purified anti-tumor poly(A) polymerase antibodies or control immunoglobulins. When the polypeptides translated from poly(A+) or poly(A-) hepatoma RNA were precipitated with immune sera, a unique [35S]methionine-labeled 35-kilodalton (kDa) protein was observed. This band was not apparent when control serum was used for the immunoprecipitation. The radiolabeled 35-kDa polypeptide was not evident when the products were incubated with highly purified tumor nuclear poly(A) polymerase prior to immunoprecipitation. Prior incubation of the translation products with bovine serum albumin instead of poly(A) polymerase had no effect on the immunoprecipitation. This 35-kDa protein was not apparent when liver poly(A+) RNA was used to direct translation. These data demonstrate that (a) the tumor enzyme is not synthesized as a precursor, (b) tumor mRNA, but not normal liver mRNA, contains detectable sequences coding for tumor-type poly(A) polymerase, and (c) poly(A) polymerase mRNA also exists as a poly(A-) population.     

Association of newly synthesized poly(A) polymerase with four distinct polypeptides

Nuclear poly(A) polymerase was isolated from [35S]methionine-labeled hepatoma McA-RH 7777 cells and subjected to DEAE-Sephadex chromatography. Flow-through and low salt wash fractions containing poly(A) polymerase activity were pooled and subjected to immunoblot analysis using anti-tumor type poly(A) polymerase antibodies and a biotinylated second antibody. The immune complex contained a single 48-kDa polypeptide band corresponding to the tumor-type enzyme. When immunoprecipitations were carried out using the same fraction and antibodies, at least five 35S-methionine-labeled proteins with approximate molecular masses of 74, 48, 35, 30, and 22 kDa were observed. Pulse-chase studies did not indicate a precursor-product relationship between the immunoprecipitated proteins. Preimmune sera did not react with poly(A) polymerase or other components in the protein complex. These data show that poly(A) polymerase exists as part of a complex with at least four other polypeptides and suggest that these polypeptides may be involved in the cleavage and/or polyadenylation reactions.     

Characterization of product RNAs synthesized in vitro by poliovirus RNA polymerase purified by chromatography on hydroxylapatite or poly(U) Sepharose.

The size of the product RNA synthesized by the poliovirus RNA polymerase and host factor was significantly affected by the type of column chromatography used to purify the polymerase. Dimer length product RNA was synthesized by the polymerase purified by chromatography on hydroxylapatite. This contrasted with the monomer length product RNA synthesized by the polymerase purified by chromatography on poly(U) Sepharose. The poly(U) Sepharose-purified polymerase was shown to contain oligo(U) that functioned as a primer. The addition of host factor to reactions containing the poly(U) Sepharose-purified polymerase significantly increased the synthesis of monomer length product RNA, in agreement with previous studies. This product RNA, however, did not immunoprecipitate with anti-VPg antibody and thus was not linked to VPg or a VPg-related protein. Thus, it was concluded that the synthesis of monomer length product RNA by the poly(U) Sepharose-purified polymerase and host factor was caused by oligo(U) priming rather than VPg priming.     

Comparison of cytosolic and nuclear poly(A) polymerases from rat liver and a hepatoma: structural and immunological properties and response to NI-type protein kinases

Poly(A) polymerases were purified from the cytosol fraction of rat liver and Morris hepatoma 3924A and compared to previously purified nuclear poly(A) polymerases. Chromatographic fractionation of the hepatoma cytosol on a DEAE-Sephadex column yielded approximately 5 times as much poly(A) polymerase as was obtained from fractionation of the liver cytosol. Hepatoma cytosol contained a single poly(A) polymerase species [48 kilodaltons (kDa)] which was indistinguishable from the hepatoma nuclear enzyme (48 kDa) on the basis of CNBr cleavage maps. Liver cytosol contained two poly(A) polymerase species (40 and 48 kDa). The CNBr cleavage patterns of these two enzymes were distinct from each other. However, the cleavage pattern of the 40-kDa enzyme was similar to that of the major liver nuclear poly(A) polymerase (36 kDa), and approximately three-fourths of the peptide fragments derived from the 48-kDa species were identical with those from the hepatoma enzymes (48 kDa). NI-type protein kinases from liver or hepatoma stimulated hepatoma nuclear and cytosolic poly(A) polymerases 4-6-fold. In contrast, the liver cytosolic 40- and 48-kDa poly(A) polymerases were stimulated only slightly or inhibited by similar units of the protein kinases. Antibodies produced in rabbits against purified hepatoma nuclear poly(A) polymerase reacted equally well with hepatoma nuclear and cytosolic enzyme but only 80% as well with the liver cytosolic 48-kDa poly(A) polymerase and not at all with liver cytosolic 40-kDa or nuclear 36-kDa enzymes. Anti-poly(A) polymerase antibodies present in the serum of a hepatoma-bearing rat reacted with hepatoma nuclear and cytosolic poly(A) polymerases to the same extent but only 40% as well with the liver cytosolic 48-kDa enzyme.(ABSTRACT TRUNCATED AT 250 WORDS)     

Identification of the subunits of GTP-binding proteins coupled to somatostatin receptors.

Somatostatin (SRIF) induces its biological effects by interacting with membrane-bound receptors that are linked to cellular effector systems via G proteins. We have studied SRIF receptor-G protein associations by solubilizing the SRIF receptor from rat brain and AtT-20 cells and immunoprecipitating the receptor-G protein complex with peptide-directed antisera against the different subunits of the G protein heterotrimer. Antiserum 8730, which selectively interacts with all Gi alpha subtypes, maximally and specifically immunoprecipitated SRIF receptor-Gi alpha complexes. To identify the subtypes of Gi alpha that are coupled to SRIF receptors, the subtype-selective antisera 3646, 1521, and 1518, which specifically interact with Gi alpha 1, Gi alpha 2, and Gi alpha 3, respectively, were used to immunoprecipitate SRIF receptor-Gi alpha complexes. Antiserum 3646 immunoprecipitated SRIF receptor-Gi alpha 1 complexes from both brain and AtT-20 cells. Antiserum 1521 immunoprecipitated Gi alpha 2 from both brain and AtT-20 cells but did not immunoprecipitate SRIF receptors from these tissues. Antiserum 1518 immunoprecipitated AtT-20 cell SRIF receptors but uncoupled brain SRIF receptor-G protein complexes. This result was confirmed with another peptide-selective antiserum, SQ, directed against Gi alpha 3. The findings from these studies indicate that Gi alpha 1 and Gi alpha 3 are coupled to SRIF receptors, whereas Gi alpha 2 is not. Even though brain and AtT-20 cell SRIF receptors were both coupled to Gi alpha, the receptors from these tissues differed in their coupling to Go alpha. Antiserum 2353, which is directed against Go alpha, immunoprecipitated SRIF receptors from AtT-20 cells, but did not immunoprecipitate or uncouple SRIF receptor-G protein complexes from rat brain. To determine the beta subunits associated with the SRIF receptor, antisera directed against G beta 36 and G beta 35 were used to immunoprecipitate SRIF receptor-G protein complexes from brain. Peptide-directed antiserum against G beta 36 selectively immunoprecipitated solubilized brain SRIF receptors. However, antiserum directed against the G beta 35 subunit did not immunoprecipitate brain SRIF receptors, suggesting that brain SRIF receptors may preferentially associate with G beta 36. In addition to coimmunoprecipitating with Gi alpha and G beta, brain SRIF receptors coimmunoprecipitated the G protein gamma subunits, G gamma 2 and G gamma 3. These results provide the first evidence that SRIF receptors are coupled to different subunits of G proteins and suggest that selectivity exists in the association of different G protein subunits with the SRIF receptor.     

Effect of poly(ADP-ribose) formation on DNA synthesis and DNA fragmentation in nuclei of rat liver and rat ascites hepatoma AH-130 cells

To elucidate the role of poly(ADP-Rib) in the nucleus, DNA synthesis and DNA fragmentation were studied in isolated nuclei of rat liver and rat ascites hepatoma AH-130 cells. Liver and hepatoma cell nuclei formed the same amount of poly(ADP-Rib) per mg of nuclear DNA from NAD. Preincubation of liver nuclei with NAD repressed DNA polymerase activity to 30% of that of the control, but preincubation of hepatoma cell nuclei with NAD did not affect DNA polymerase activity. It was also found that incubation of liver nuclei with NAD prevented the fragmentation of nuclear DNA which occurred without NAD. Incubation of hepatoma cell nuclei with or without NAD did not result in fragmentation of DNA. The role of endonuclease in primer formation for DNA synthesis is discussed.     

Expression of neuronal acetylcholine receptor polypeptidesin vitro

1. Translation of poly(A) RNA extracted from the nervous tissue of locusts in a reticulocyte lysate system led to polypeptides with a broad spectrum of molecular weights. 2. Using anti-locust acetylcholine receptor (AChR) antisera, polypeptides with a molecular weight of about 50,000 were immunoprecipitated. These peptides comprised about 0.3% of the total translation products. 3. Cotranslational incubation with pancreatic rough microsomes resulted in a glycosylated 60,000-dalton immunoprecipitate. 4. Density-gradient analysis of in vitro synthesized and glycosylated receptor polypeptides indicated that no assembly of subunits had taken the place under the in vitro conditions.     

Purification and characterization of a nuclear protein kinase from rat liver and a hepatoma that is capable of activating poly(A) polymerase

The cyclic nucleotide-independent protein kinase which is separated from poly(A) polymerase during its purification from nuclei of rat liver and Morris hepatoma 3924A was purified essentially to homogeneity. Liver nuclear poly(A) polymerase was dissociated from protein kinase by phosphocellulose column chromatography. In contrast, protein kinase copurified with the hepatoma poly(A) polymerase on the phosphocellulose column. Neither liver nor hepatoma kinase was stimulated by spermine or inhibited by heparin. These enzymes did not utilize GTP as phosphoryl donor, or histones or tyrosine-containing [Val5]-angiotensin II as phosphoryl acceptors. The apparent Km with respect to ATP was similar for the liver (4.7 microM) and hepatoma (11 microM) kinases, and the apparent Km with respect to casein was identical (0.6 microgram/microliter) for these enzymes. Both enzymes were capable of phosphorylating poly(A) polymerase and stimulating both tumor and liver poly(A) polymerase activity. However, in addition to their different chromatographic properties, the two kinases differed in molecular weight (liver, 37,000; hepatoma, 56,000), in their response to various divalent metal ions, and in their ability to phosphorylate hepatoma poly(A) polymerase (Km 7.9 and 30 microgram/microliter for liver and hepatoma enzymes, respectively). These latter characteristics distinguished the liver and hepatoma protein kinases from each other as well as from the previously described NI protein kinase.     

Eukaryotic small ribonucleoproteins. Anti-La human autoantibodies react with U1 RNA-protein complexes

Anti-La sera from patients with autoimmune disorders precipitate a set of nuclear and cytoplasmic small RNA-protein complexes. Up to now, it has been thought that the La antigen is associated only with RNAs transcribed by RNA polymerase III, including precursors of tRNA and 5 S ribosomal RNA. Here we report that anti-La sera also react with ribonucleoprotein particles containing small nuclear RNA U1, which is transcribed by RNA polymerase II. Anti-La sera from 12 out of 12 patients tested were found to precipitate U1 RNA-protein complexes from HeLa cell nuclear extracts, under conditions where nonimmune sera do not. Ribonucleoprotein particles containing a second small nuclear RNA, U2, do not react appreciably with anti-La sera although they are present in HeLa cell nuclei at the same concentration as U1 RNA. Anti-La sera also react with U1 RNA-protein complexes in mouse and frog cells, but not in Drosophila or Chironomus, two organisms which lack the La antigen. Hybridization of cloned U1 DNA with anti-La-reactive RNA from HeLa cell nuclear extracts reveals mature U1 RNA, whereas anti-La-reactive cytoplasmic RNA contains a series of hybridizing bands that represent molecules 1-7 nucleotides longer than U1 and which may include precursors of nuclear U1 RNA (Madore, S. J., Wieben, E. D., and Pederson, T. (1984) J. Cell Biol., 188-192). Pulse-chase experiments suggest that the association of La antigenicity with these cytoplasmic U1 RNA molecules is transient. These results are discussed in relation to the presence of uridylate-rich sequences in the 3' termini of U1 RNA precursors and mature U1 RNA, which are similar to La antigen binding sites in several RNAs transcribed by RNA polymerase III.     

Structurally and immunologically distinct poly(A) polymerases in rat liver. Occurrence of a tumor-type enzyme in normal liver

Poly(A) polymerases purified from rat liver nuclei consisted of two distinct species, a predominant enzyme of Mr = 38,000 and a minor one of Mr = 48,000. Prior to extensive purification, the minor enzyme constituted approximately 1% of the total liver poly(A) polymerase. Poly(A) polymerase purified from a rat tumor, Morris hepatoma 3924A, was comprised of a single species of Mr = 48,000 which was identical to the minor liver enzyme with respect to chromatographic and immunological characteristics. Gel filtration on Sephacryl S-200 using 0.3 M NaCl for elution showed that the major liver poly(A) polymerase had a molecular weight of 156,000, which corresponded to a tetramer of the 38-kDa polypeptide, whereas the hepatoma and minor liver 48-kDa species existed as dimers with a molecular weight of 96,000. Fractionation by Sephacryl S-200 resulted in complete loss of both liver poly(A) polymerase activities which could be restored by exogenous N1-type protein kinase. Following CNBr cleavage, the 48-kDa poly(A) polymerase from liver and hepatoma exhibited nearly identical peptide maps which were distinct from that of the major liver enzyme (38 kDa). Antibodies raised against tumor poly(A) polymerase reacted with the 48-kDa polypeptide but not with the 38-kDa liver enzyme. Immune complex formation was observed between seven of the eight CNBr cleavage products derived from the 48-kDa polypeptide of both liver and hepatoma. It is concluded that distinct genes in rat liver code for two structurally and immunologically unique nuclear poly(A) polymerases, one of which is identical to the enzyme from the hepatoma.     

Novel human autoantibodies reactive with 5'-terminal trimethylguanosine cap structures of U small nuclear RNA.

A class of RNA-containing particles, U small nuclear/nucleolar ribonucleoprotein particles (U snRNP), are well known to be targets for sera from patients with various autoimmune diseases. In the most cases the protein components carry the antigenic determinants. We have identified serum autoantibodies from three patients with systemic sclerosis that were directed against U1-U5 snRNA by immunoprecipitation of deproteinized 32PO4 labeled HeLa cell total RNA. By competitive radioimmunoprecipitation assays, an experimentally induced anti-2,2,7-trimethylguanosine (TMG) cap structure mAb inhibited the reaction of these antisera. In addition, IgG isolated from the antisera inhibited the anti-TMG mAb reaction to the U snRNA. Furthermore, a structural analog, 7-methylguanosine-triphosphate, competitively inhibited the reaction of the antisera to the U snRNA. Thus we concluded that the TMG cap structure of the U snRNA could be a target for serum autoantibodies.     

Hepatoma-associated nuclear matrix nonhistone antigens

Polyclonal antibodies generated against a group of high molecular weight nonhistone proteins from Morris hepatoma 7777 were used in immunological studies of hepatoma-associated nonhistone proteins in rat and hamster. We revealed the presence of cross-reactive antigens in rat Morris hepatomas 7777 and 8994, and in hamster Kirkman-Robbins hepatoma, but not in normal rat or hamster livers. These specific nonhistone proteins were found to be preferentially localized in the nuclear matrix of rat Morris hepatoma 7777 as well as hamster Kirkman-Robbins hepatoma.     

Hepatoma-associated non-histone proteins are phosphoproteins preferentially localized in nuclear matrix

1. High molecular weight non-histone proteins (NHP) were isolated from Morris hepatoma 7777 by Sephadex G-100, S-200 chromatography. 2. Specific polyclonal antibodies were raised against these NHP in rabbits. These antibodies recognized specific NHP components present in Morris hepatoma 7777 and 8994, but not in normal rat liver. Hepatoma-associated antigens are phosphoproteins. 3. Immunologically specific NHP of Morris hepatoma are intensively concentrated in nuclear matrix fraction.     

Newly identified U4/U6 snRNP-binding proteins by serum autoantibodies from a patient with systemic sclerosis.

We found serum autoantibodies directed against the proteins binding exclusively to U4/U6 of Sm small nuclear ribonucleoprotein particle (snRNP) in serum from a patient (MaS) with systemic sclerosis. Their specificity, called anti-MaS, is distinct from that of known antibodies against U snRNP. The U4 and U6 small nuclear RNA from a 32P-labeled HeLa cell extract and five proteins with Mr 150,000, 120,000, 80,000, 36,000, and 34,000, in addition to Sm core proteins (B, B', D, E, F, and G) from an [35S] methionine-labeled extract, were immunoprecipitated by anti-MaS in isotonic solution. However, the Sm core proteins and U4 and U6 small nuclear RNA were separated from the protein-A-Sepharose facilitated MaS immunoprecipitate by incubation in a solution containing 500 mM NaCl. In immunoblots, anti-MaS antibodies reacted with one protein of Mr 150,000 from a HeLa cell nuclear extract that was fractionated by SDS-PAGE and transferred to a nitrocellulose sheet. The monospecific immunoaffinity purified antibody eluted from the immunoblot band immunoprecipitated U4 and U6 small nuclear RNA and reblotted the protein with Mr 150,000. These data indicate that anti-MaS antibodies recognize at least one antigenic protein that binds exclusively to the U4/U6 snRNP.     

Biosynthesis of rat insulin-like growth factor II. I. Immunochemical demonstration of a approximately 20-kilodalton biosynthetic precursor of rat insulin-like growth factor II in metabolically labeled BRL-3A rat liver cells

BRL-3A rat liver cells synthesize mature 7484-dalton rat insulin-like growth factor II (rIGF-II) as a approximately 22-kDa precursor, presumably prepro-rIGF-II. In the present study, we have biosynthetically labeled intact BRL-3A cells with [35S]cysteine and immunoprecipitated cell lysates and media with antisera to rIGF-II. A approximately 20-kDa protein was identified in immunoprecipitates of cell lysates having properties consistent with pro-rIGF-II. The approximately 20-kDa protein is precipitated by immune sera but not by nonimmune serum. Its immunoprecipitation is specifically inhibited by unlabeled rIGF-II but not by insulin. It is not precipitated from labeled lysates of a subclone of BRL-3A cells (BRL-3A2) that does not synthesize rIGF-II. The approximately 20-kDa protein is rapidly labeled intracellularly (10 min) but is not detected in BRL-3A media. In pulse-chase experiments, radioactivity in the approximately 20-kDa protein disappears during the chase and appears, at later times, in specifically immunoprecipitated approximately 19-, approximately 10-, approximately 8-, and approximately 7-kDa proteins in media and, to a limited extent, intracellularly. A protein with electrophoretic mobility identical to that of the approximately 20-kDa protein observed in cell lysates is immunoprecipitated from 35S-proteins whose synthesis is directed by BRL-3A RNA in a reticulocyte lysate cell-free translation system supplemented with microsomal membranes, and presumably arises by cotranslational removal of the signal peptide from approximately 22-kDa prepro-rIGF-II. Processing of the approximately 20-kDa protein in intact BRL-3A cells to intermediate and mature rIGF-II species appears to occur at the time of secretion and/or shortly thereafter, with the different forms appearing at approximately the same time.