Alternative translation initiation augments the human mitochondrial proteome.

Alternative translation initiation (ATI) is a mechanism of producing multiple proteins from a single transcript, which in some cases regulates trafficking of proteins to different cellular compartments, including mitochondria. Application of a genome-wide computational screen predicts a cryptic mitochondrial targeting signal for 126 proteins in mouse and man that is revealed when an AUG codon located downstream from the canonical initiator methionine codon is used as a translation start site, which we term downstream ATI (dATI). Experimental evidence in support of dATI is provided by immunoblotting of endogenous truncated proteins enriched in mitochondrial cell fractions or of co-localization with mitochondria using immunocytochemistry. More detailed cellular localization studies establish mitochondrial targeting of a member of the cytosolic poly(A) binding protein family, PABPC5, and of the RNA/DNA helicase PIF1α. The mitochondrial isoform of PABPC5 co-immunoprecipitates with the mitochondrial poly(A) polymerase, and is markedly reduced in abundance when mitochondrial DNA and RNA are depleted, suggesting it plays a role in RNA metabolism in the organelle. Like PABPC5 and PIF1α, most of the candidates identified by the screen are not currently annotated as mitochondrial proteins, and so dATI expands the human mitochondrial proteome.

Preliminary crystallographic analysis of a polyadenylate synthase from Megavirus.

Megavirus chilensis, a close relative of the Mimivirus giant virus, is also the most complex virus sequenced to date, with a 1.26 Mb double-stranded DNA genome encoding 1120 genes. The two viruses share common regulatory elements such as a peculiar palindrome governing the termination/polyadenylation of viral transcripts. They also share a predicted polyadenylate synthase that presents a higher than average percentage of residue conservation. The Megavirus enzyme Mg561 was overexpressed in Escherichia coli, purified and crystallized. A 2.24 Šresolution MAD data set was recorded from a single crystal on the ID29 beamline at the ESRF.

Identification and characterization of nuclear non-canonical poly(A) polymerases from Trypanosoma brucei.

Regulation of nuclear genome expression in Trypanosoma brucei is critical for this protozoan parasite's successful transition between its vertebrate and invertebrate host environments. The canonical eukaryotic circuits such as modulation of transcription initiation, mRNA splicing and polyadenylation appear to be nearly non-existent in T. brucei suggesting that the transcriptome is primarily defined by mRNA turnover. Our previous work has highlighted sequence similarities between terminal RNA uridylyl transferases (TUTases) and non-canonical poly(A) polymerases, which are widely implicated in regulating nuclear, cytoplasmic and organellar RNA decay throughout the eukaryotic lineage. Here, we have continued characterization of TUTase-like proteins in T. brucei and identified two nuclear non-canonical poly(A) polymerases (ncPAPs). The 82kDa TbncPAP1 is essential for viability of procyclic and bloodstream forms of T. brucei. Similar to Trf4/5 proteins from budding yeast, TbncPAP1 requires protein cofactor(s) to exert poly(A) polymerase activity in vitro. The recombinant 54kDa TbncPAP2 showed a PAP activity as an individual polypeptide. Proteomic analysis of the TbncPAP1 interactions demonstrated its association with Mtr4 RNA helicase and several RNA binding proteins, including a potential ortholog of Air1p/2p proteins, which indicates the presence of a stable TRAMP-like complex in trypanosomes. Our findings suggest that TbncPAP1 may be a "quality control" nuclear PAP involved in targeting aberrant or anti-sense transcripts for degradation by the 3'-exosome. Such mechanisms are likely to play a major role in alleviating promiscuity of the transcriptional machinery.

Multiple forms of poly(A) polymerases purified from HeLa cells function in specific mRNA 3'-end formation.

Poly(A) polymerases (PAPs) from HeLa cell cytoplasmic and nuclear fractions were extensively purified by using a combination of fast protein liquid chromatography and standard chromatographic methods. Several forms of the enzyme were identified, two from the nuclear fraction (NE PAPs I and II) and one from the cytoplasmic fraction (S100 PAP). NE PAP I had chromatographic properties similar to those of S100 PAP, and both enzymes displayed higher activities in the presence of Mn2+ than in the presence of Mg2+, whereas NE PAP II was chromatographically distinct and had approximately equal levels of activity in the presence of Mn2+ and Mg2+. Each of the enzymes, when mixed with other nuclear fractions containing cleavage or specificity factors, was able to reconstitute efficient cleavage and polyadenylation of pre-mRNAs containing an AAUAAA sequence element. The PAPs alone, however, showed no preference for precursors containing an intact AAUAAA sequence over a mutated one, providing further evidence that the PAPs have no intrinsic ability to recognize poly(A) addition sites. Two additional properties of the three enzymes suggest that they are related: sedimentation in glycerol density gradients indicated that the native size of each enzyme is approximately 50 to 60 kilodaltons, and antibodies against a rat hepatoma PAP inhibited the ability of each enzyme to function in AAUAAA-dependent polyadenylation.

Cloning and characterization of a Xenopus poly(A) polymerase.

During oocyte maturation and early embryogenesis in Xenopus laevis, the translation of several mRNAs is regulated by cytoplasmic poly(A) elongation, a reaction catalyzed by poly(A) polymerase (PAP). We have cloned, sequenced, and examined several biochemical properties of a Xenopus PAP. This protein is 87% identical to the amino-terminal portion of bovine PAP, which catalyzes the nuclear polyadenylation reaction, but lacks a large region of the corresponding carboxy terminus, which contains the nuclear localization signal. When injected into oocytes, the Xenopus PAP remains concentrated in the cytoplasm, suggesting that it is a specifically cytoplasmic enzyme. Oocytes contain several PAP mRNA-related transcripts, and the levels of at least the one encoding the putative cytoplasmic enzyme are relatively constant in oocytes and early embryos but decline after blastulation. When expressed in bacteria and purified by affinity and MonoQ-Sepharose chromatography, the protein has enzymatic activity and adds poly(A) to a model substrate. Importantly, affinity-purified antibodies directed against Xenopus PAP inhibit cytoplasmic polyadenylation in egg extracts. These data suggest that the PAP described here could participate in cytoplasmic polyadenylation during Xenopus oocyte maturation.

Separation of factors required for cleavage and polyadenylation of yeast pre-mRNA.

Cleavage and polyadenylation of yeast precursor RNA require at least four functionally distinct factors (cleavage factor I [CF I], CF II, polyadenylation factor I [PF I], and poly(A) polymerase [PAP]) obtained from yeast whole cell extract. Cleavage of precursor occurs upon combination of the CF I and CF II fractions. The cleavage reaction proceeds in the absence of PAP or PF I. The cleavage factors exhibit low but detectable activity without exogenous ATP but are stimulated when this cofactor is included in the reaction. Cleavage by CF I and CF II is dependent on the presence of a (UA)6 sequence upstream of the GAL7 poly(A) site. The factors will also efficiently cleave precursor with the CYC1 poly(A) site. This RNA does not contain a UA repeat, and processing at this site is thought to be directed by a UAG...UAUGUA-type motif. Specific polyadenylation of a precleaved GAL7 RNA requires CF I, PF I, and a crude fraction containing PAP activity. The PAP fraction can be replaced by recombinant PAP, indicating that this enzyme is the only factor in this fraction needed for the reconstituted reaction. The poly(A) addition step is also dependent on the UA repeat. Since CF I is the only factor necessary for both cleavage and poly(A) addition, it is likely that this fraction contains a component which recognizes processing signals located upstream of the poly(A) site. The initial separation of processing factors in yeast cells suggests both interesting differences from and similarities to the mammalian system.

Polyadenylic acid polymerase activity in normal and leukemic human leukocytes.

Soluble polyadenylic acid [poly(A)] polymerase and poly(A) nucleases content of normal human blood lymphocytes and leukemic blood cell populations was determined. Blood lymphocytes from seven normal individuals were used as controls. Leukemic cells were obtained from 69 patients with various types of acute and chronic leukemias. Chronic lymphocytic leukemias presented poly(A) polymerase values with a mean of 9 +/- 4 (S.D.). Although most of the chronic lymphocytic leukemia cases presented poly(A) polymerase activities similar to those of normal lymphocytes (3 +/- 3), a small number fell into the specific activity values of acute leukemias, which were significantly higher and covered a wider range. The mean values for acute myeloblastic, acute monoblastic, and acute lymphoblastic leukemias were 53 +/- 50, 21 +/- 8, and 29 +/- 14, respectively. A statistically significant difference was found between chronic and acute leukemias (p less than 0.01). The observed differences in poly(A) polymerase levels of acute lymphoblastic leukemia versus chronic lymphocytic leukemia persisted after fractionation of the crude extracts and, furthermore, they could not be attributed to differences in the levels of poly(A)-degrading enzymes [poly(A) endo- and exonucleases]. Fractionation of leukemic extracts on Sephadex G-75 revealed two molecular forms of poly(A) polymerase activity.

Poly(A) polymerases of rat liver nuclei. Purification and specificity.

Two poly(A) polymerases were isolated from rat liver nuclei and purified more than one thousand times by ion exchange chromatography on DEAE-Sephadex and phosphocellulose columns as well as affinity chromatography on a chromosomal RNA-Sepharose column. One of the two enzymes is bound to chromatin and uses as primer chromosomal RNA, while the second one is localized in the nucleoplasm and uses as primer poly(A) and hnRNA isolated from chromatin. The two enzymes seem to participate in the polyadenylation of chromosomal RNA in vitro, by a coupled mechanism. According to this mechanism, the chromatin bound enzyme adds 120-130 adenosine nucleotides to chromosomal RNA and consequently the nucleoplasmic enzyme completes the poly-adenylation by adding 80-90 more AMP units to the polyadenylated end of chromosomal RNA.

A kinetic and structural characterization of adenosine-5'-triphosphate: ribonucleic acid adenylyltransferase from Pseudomonas putida.

A catalytic and structural study of ATP:RNA adenylyltransferase (EC 2.7.7.19) from the particulate fraction of Pseudomonas putida was made. During the large-scale purification of this enzyme, designated adenylyltransferase B, a previously undetected ATP-incorporating activity, designated adenylyltransferase A, was observed. Adenylyltransferases A and B were indistinguishable catalytically; however, they differed in their chromatographic and sedimentation properties. Adenylyltransferases A and B were resolved by phosphocellulose, by poly (U)-Sepharose and by Bio-Gel P-100 chromatographies. Adenylytransferase A was determined to have a sedimentation coefficient (S020,w) of 9.3 S and B of 4.3 S. The molecular weight of adenylyltransferase A was estimated to be 185000 and that of adenylyltransferase B to be 50000-60000. Apparently, adenylyltransferase A was generated from adenylyltransferase B during the purification. The AMP incorporation catalyzed by adenylyltransferases A and B was inhibited by two derivatives of the antibiotic rifamycin, AF/013 (50% at 5 mug/ml) and AF/DNFI (50% at 10 mug/ml). The 5'-triphosphate derivative (3'-dATP) of the drug cordycepin (3'-deoxyadenosine/ was a competitive inhibitor with ATP for both adenylyltransferases. The Ki for 3'-deoxyadenosine 5'-triphosphate was 6 - 10(-4)--10 - 10(-4) M, while the Km for ATP was 1 - 10(-4)--2 - 10(-4) M. Several other anaolgs of ATP, 2'-deoxyadenosine 5' triphosphate, 2'-O-methyl ATP, or the fluorescent 3-beta-D-ribofuranosylimidazo [2,1-i] purien 5'-triphosphate did not affect the activity of adenylyltransferase A or B. Poly(U) and poly(dT) were competitive inhibitors of the ribosomal RNA-primed polymerization reaction. The Ki for poly(U) or poly(dT), in terms of nucleotide phosphate, was 4 - 10-6)--10 - 10(-6) M for adenylyltransferases A and B, compared to 2 - 10(-4)--4 - 10(-4) M for the Km of ribosomal RNA. The inhibition was a result of the competition between the non-priming poly(U), or poly(dT), and ribosomal RNA for the primer binding site on the enzyme.

Polyadenylate polymerase from cytoplasm and nuclei of N.I.H.-Swiss mouse embryos.

Poly (A) polymerase activity from cytoplasm and nuclei of 12-16-day-old mouse embryos has been partially purified by (NH4)2SO4 fractionation, DEAE-cellulose, phosphocellulose and tRNA-Sepharose affinity chromatography, and their properties have been compared. The nuclear and cytoplasmic enzymes exhibit similar chromatographic elution profiles, and similar biochemical and physical properties. Poly(A) polymerase has an absolute requirement for a divalent cation, ATP and an oligo- or polyribonucleotide primer. With tRNA, the divalent salt concentrations for optimum enzyme activity are 1 mM MnCl2 or 10 mM MgCl2. The enzyme activity with MnCl2 is 10-15-fold higher than that with MgCl2. The molecular weight of the native enzyme is about 65 000 and its sedimentation coefficient is around 4.5 S. The average chain length synthesized by the enzyme is between 10 and 13 nucleotides. The inhibitors of RNA polymerase do not affect poly (A) polymerase activity; however, some synthetic rifamycin SV derivatives are potent inhibitors of this enzyme.

Purification and characterization of full-length mammalian poly(A) polymerase.

Bovine poly(A) polymerase was purified from overexpressing strains of Escherichia coli and from Spodoptera frugiperda Sf21 cells infected with a recombinant baculovirus. The E. coli-expressed enzyme had an apparent molecular mass of 85 kDa in SDS gels, as anticipated from the cDNA sequence. Poly(A) polymerase from insect cells consisted of several species with higher apparent molecular weights due to phosphorylation. The two preparations showed minor differences in their catalytic properties. The insect cell-expressed enzyme had a 5-fold higher Km for the primer in a nonspecific Mn(2+)-dependent polyadenylation reaction and a lower activity in specific AAUAAA-dependent polyadenylation and generated shorter poly(A) tails during the processive phase of polyadenylation. Both recombinant poly(A) polymerases stimulated 3'-cleavage of the SV40 late mRNA precursor. Neither preparation contained ATPase or poly(A) degrading activity. The enzyme polymerized adenosine 5'-O-(1-thiotriphosphate), SP-diastereomer, with inversion of configuration. Thus, poly(A) synthesis proceeds via an SN2-in-line mechanism without covalent intermediate.

Purification and characterization of a poly(A) polymerase from beef liver nuclei.

Poly(A) polymerase [EC 2.7.7.19] was highly purified from beef liver nuclei by the use of column chromatographies on heparin-Sepharose 4B and Blue Dextran-Sepharose 4B. The purified enzyme showed one major protein band of the molecular weight of 57,000 in SDS polyacrylamide gel electrophoresis, which agreed with the molecular weight estimated from glycerol gradient centrifugation. The enzyme required the presence of Mn2+ for its activity but was almost completely inactive with Mg2+. It incorporated specifically ATP into polynucleotide as a sole substrate. The enzyme activity dependend entirely on the addition of exogenous polynucleotide primer. It showed certain selectivity for the primers. The most effective among the tested polynucleotides was a short poly(A), for which the Km of the enzyme was shown to be 7 microM. Poly(G, U) and short poly(U) also primed the reaction, but tRNA, phage RNA, poly(G), and poly(C) were inactive. Based on observed specificity for the primer, the role of this enzyme in the cell nuclei was discussed. Digestion of the reaction product of this enzyme by two specific exonucleases, snake venom and spleen phosphodiesterases, suggested that this enzyme catalyzed the covalent bonding of the substrate to the 3' terminus of the primer as in the manner expected for in vivo polyadenylation.

Characterization of polyriboadenylate polymerase from Tetrahymena pyriformis.

Poly(A) polymerase [polyadenylate nucleotidyltransferase, EC 2.7.7.19] was extracted from Tetrahymena pyriformis. The enzyme was demonstrated to be present in three forms by column chromatography on DEAE-cellulose, and they were termed poly(A) polymerase Ia, Ib, and II in order of increasing affinity to the column. The properties of enzymes Ia and Ib were similar except that Ia utilizes poly(A) as a primer rather efficiently. Enzyme II differed from enzymes Ia and Ib not only in elution profile on DEAE-cellulose column chromatography but also in pH and temperature preferences, molecular weight, requirement for divalent cations, sensitivity to salts at high ionic strength, optimal primer concentration, and subcellular localization. The molecular weights of enzymes Ia and Ib measured by gel filtration were both 43,000, and that of enzyme II was 95,000. All three enzymes required Mn2+ for maximal activity; Mg2+ could replace Mn2+ in the reaction of enzyme II, but only partially. In the presence of 0.1 M ammonium sulfate the activities of enzymes Ia and Ib were both completely inhibited, whereas enzyme II still showed 42% of its original activity. These findings suggest that there are two distinct types of poly(A) polymerase in Tetrahymena pyriformis.

Simultaneous presence of terminal adenylyl, cytidylyl, guanylyl, and uridylyl transferase in healthy tomato leaf tissue: separation from RNA-dependent RNA polymerase and characterization of the terminal transferases.

The presence of terminal nucleotidyl transferase activities catalyzing the addition of AMP, CMP, GMP, and UMP residues to the 3' ends of oligonucleotide primers was detected in healthy tomato plants. These enzyme activities copurify with RNA-dependent RNA polymerase during the initial stages of purification. Their separation from RNA-dependent RNA polymerase is finally achieved by DEAE chromatography: terminal transferase activities are retained on DEAE while RNA-dependent RNA polymerase does not bind in the presence of 20 mM MgCl2. Elution by a linear gradient of 0 to 400 mM NH4Cl releases all four terminal transferase activities from the DEAE column at a concentration of 270 mM NH4Cl, thus suggesting that they may belong to one enzyme molecule; this question, however, needs further clarification. The enzyme activities are completely dependent on the presence of an RNA primer and are strongly influenced by its base composition as well as its chain length. Characterization of the respective reaction products by electrophoresis on 15% polyacrylamide sequencing gels reveals striking differences as to the number of nucleotides added to a given primer. In the case of UMP transfer to U8 or A8 and in the case of GMP transfer to A8 only 1 to 6 nucleoside monophosphates are added to the 3' terminus of the oligonucleotide primer, whereas in the case of AMP transfer to A8 or U8, the CMP transfer to A8, and the GMP transfer to U8, longer chains of minimally 30 nucleotides are added to the respective primer. After gradient elution from DEAE the transferase preparation displays no nucleolytic activity when incubated in the presence of 3H-labelled ribosomal RNA or [3H]poly(A) X poly(U). Only in the case of [3H]poly(A) and [3H]poly(U) or [3H]poly(C) 10 to 15% of the radioactivity is transferred to acid-soluble counts.

Drug action on poly(A) polymerase activity and isoforms during U937 cell apoptosis.

The enzyme poly(A) polymerase (PAP; EC 2.7.7.19) catalyzes the polyadenylation of mRNAs. It's activity levels and isoforms vary within the cell cycle (31) and apoptosis (34). The direct effect of most anticancer drugs is cell damage (DNA and RNA synthesis inhibition, DNA breaks and/or cell cycle aberrations), which then triggers signaling pathways that activate apoptosis and eventually lead to regulated cell death. The experiments described here concern the chemotherapeutic agents, interferon (IFN) and 5-fluorouracil (5-FU), and their action on U937 cells, alone or in various combinations, resulting in the commitment of cell apoptosis, as observed by the appearance of DNA fragmentation. Furthermore, examination of U937 cell apoptotic trend in parallel with PAP activity measurements and isoforms detection by immunoblotting, revealed both partial enzyme inactivation and dephosphorylation, in particular after the combined drug action of 5-FU and IFN on U937 cells. Our work on chemotherapeutic drug action at the level of mRNA polyadenylation may contribute to new insights into the mechanism of cell apoptosis, as well as provide information on mRNA poly(A) tail formation, removal and function.

Large-scale production and purification of recombinant protein from an insect cell/baculovirus system in Erlenmeyer flasks: application to the chicken poly(ADP-ribose) polymerase catalytic domain.

A simple and inexpensive shaker/Erlenmeyer flask system for large-scale cultivation of insect cells is described and compared to a commercial spinner system. On the basis of maximum cell density, average population doubling time and overproduction of recombinant protein, a better result was obtained with a simpler and less expensive bioreactor consisting of Erlenmeyer flasks and an ordinary shaker waterbath. Routinely, about 90 mg of pure poly(ADP-ribose) polymerase catalytic domain was obtained for a total of 3 x 10(9) infected cells in three liters of culture.

Auxin regulated poly(A)polymerase activity in Cicer arietinum epicotyls.

There was a linear increase in poly (A+) polymerase activity in the C. arietinum epicotyls during germination. Six-day-old auxin treated seedlings showed about 3-4 fold stimulation in enzyme activity, accompanied with 3- fold rise in the relative abundance of poly (A+) RNA levels. Actinomycin D, cycloheximide, cordycepin and amino acid analogues caused dramatic decline in poly (A+) polymerase as well as poly (A+) RNA levels. It seems that auxin induced a de novo synthesis of this enzyme.

[Polyadenylation of influenza virion RNA in an in vitro system]. / Poliadenilirovanie virionnoi RNK grippa v sisteme in vitro.

A method for isolation of terminal polyriboadenylate transferase from E. coli cells (MPE 600) is presented. The specific activity of the enzyme yield at the terminal stage was 933 units per 1 mg of protein. Analysis of polyadenylated in vitro virion RNA of influenza virus A/USSR/90/17 strain in polyacrylamideagarose gel (2.2%-0.6%) in the presence of 6 M urea showed all the 8 fragments of genome RNA to be adenylated, their sizes being retained with regard to the distribution in gel of the initial RNA fragments. In vitro polyadenylated virion RNA was an effective matrix in reverse transcription reaction with RNA-dependent DNA-polymerase using oligo (dT) as a primer. Complementary DNAs obtained in this way may be the starting material for synthesis of double-stranded DNAs and subsequent construction of recombinant DNAs containing influenza virus genetic information.