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Isolation and Expression of Cdna Clones Encoding Mammalian Poly(A) Polymerase

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Isolation and Expression of Cdna Clones Encoding Mammalian Poly(A) Polymerase The EMBO Journal vol.10 no.13 pp.4251 -4257, 1991 Isolation and expression of cDNA clones encoding mammalian poly(A) polymerase Elmar Wahle, Georges Martin, Emile Schiltz1 et al., 1989; Gilmartin and Nevins, 1989) and depends on and Walter Keller2 a second sequence element downstream of the cleavage site (reviewed by Proudfoot, 1991). Biozentrum der Universitat Basel, Abteilung Zellbiologie, Poly(A) polymerase has been purified to homogeneity Klingelbergstrasse 70, CH4056 Basel, Switzerland and 'Institut fiir from calf thymus as a protein of 57-60 kDa (Tsiapalis et al., Organische Chemie und Biochemie der Universitat Freiburg, Albertstrasse 21, D-7800 Freiburg, FRG 1975; Wahle, 1991a). The enzyme by itself does not recognize the polyadenylation signal AAUAAA. In fact, it 2To whom correspondence should be addressed has a poor affinity for any RNA substrate and only a slight Communicated by W.Keller preference for poly(A) (Wahle, 1991a). The addition of Mn2+ to the reaction nonspecifically increases the affinity cDNA clones encoding mammalian poly(A) polymerase of the enzyme for RNA primers and permits efficient were isolated with probes generated by the polymerase polyadenylation independent of AAUAAA and CPF chain reaction based on amino acid sequences derived (Tsiapalis et al., 1975; Christofori and Keller, 1988; from the purified enzyme. A bovine cDNA clone was Takagaki et al., 1988; Wahle, 1991a). obtained encoding a protein of 82 kDa. Expression in We have used amino acid sequences derived from the Escherichia coli resulted in the appearance of a poly(A) purified enzyme to isolate cDNA clones of poly(A) polymerase activity that was dependent on the addition polymerase. Expression of a clone in Escherichia coli of the purified specificity factor CPF and the presence confirmed that it encoded poly(A) polymerase functional in of the polyadenylation signal AAUAAA in the RNA the AAUAAA-dependent and CPF-mediated polyadenylation substrate. The activity copurified with a polypeptide of of RNA. the expected size. A second class of cDNAs encoded a polypeptide of 43 kDa which was closely related to the N-terminal half of the 82 kDa protein. Northern blots Results showed two mRNAs of 4.2 and 2.4 kb that probably Isolation of a cDNA clone encoding poly(A) correspond to the two classes of cDNAs, as well as a third polymerase band of 1.3 kb. The sequence of the N-terminal half of Amino acid sequences were obtained from the N-terminus bovine poly(A) polymerase is 47% identical with the as well as from three tryptic peptides of purified poly(A) amino acid sequence of the corresponding part of yeast polymerase (Figure 1). DNA oligonucleotides to be used as poly(A) polymerase. Homologies to other proteins are of primers for the polymerase chain reaction (PCR) were uncertain significance. synthesized based on some of these amino acid sequences Key words: cDNA cloning/polyadenylation/poly(A) poly- (Figure 1 and Materials and methods). The templates used merase/mRNA processing for PCR were obtained by reverse transcription of calf thymus RNA, primed either by oligo(dT) or by the same DNA oligonucleotide that was used as a PCR primer (see Introduction Materials and methods). A DNA fragment of 700 nucleotides was obtained, among others, in a PCR reaction using primers The polyadenylate tails present on almost all eukaryotic 1 and 4. After gel purification, this fragment could be messenger RNAs are added post-transcriptionally by a reamplified with primers 2 and 4. Direct sequencing of the multicomponent machinery (reviewed by Manley, 1988; Wickens, 1990). Synthesis of the poly(A) tail is carried out Peptide: Sequence: by poly(A) polymerase with the help of the cleavage and 1 polyadenylation factor, CPF (Christofori and Keller, 1988, 2'p 1989; Takagaki et al., 1988, 1989; Gilmartin and Nevins, N-terminus PFPVTTQGSQQTQPXQKXYG 1989; Wahle, 1991a; Bienroth et al., 1991). CPF [also called 4 SF (Takagaki et al., 1989) or PF2 (Gilmartin and Nevins, 101 TDEILHLtPNIDNFRLTLRAIKLXAK 1989)] binds to the essential polyadenylation signal AAUAAA which is located in the RNA 10-30 nucleotides 96 THNIYSNILGFLGGVSXAMLVAR upstream of the polyadenylation site (Gilmartin and Nevins, 66 QRLEWVGLVESK 1989; Bardwell et al., 1991; Keller et al., 1991). Elongation of the growing tail is assisted by a poly(A) binding protein Fig. 1. Peptide sequences of purified poly(A) polymerase. Sequences (Wahle, 199 lb; Gershon et al., 1991). The polyadenylation of the N-terminus and three internal peptides (arbitrary numbering) are of the presented in the single letter code in their order of appearance in the reaction is preceded by endonucleolytic cleavage predicted protein sequence (see Figure 2). X, unidentified amino acid. primary transcript at the polyadenylation site. This cleavage The primers used for PCR amplification of cDNA fragments are reaction requires at least two factors in addition to poly(A) indicated as arrows pointing in the 5' to 3' direction (for details, see polymerase and CPF (Christofori and Keller, 1988; Takagaki Materials and methods). ©J Oxford University Press 4251 E.Wahle et al. PCR product with primer 2 revealed a nucleotide sequence 82.4 kDa. The N-terminus of the predicted amino acid downstream of the primer that encoded six amino acids sequence was identical with the N-terminal sequence obtained known from the sequencing of the purified protein (data not from purified poly(A) polymerase except for the absence of shown). Correct products, identified by similar procedures, the initiating methionine in the protein. The predicted were also obtained with other primer combinations (data not sequence also contained all internal peptide sequences shown). derived from the purified protein. The 700 bp fragment obtained with primers 2 and 4 was used as a probe to screen two cDNA libraries, derived from Expression of poly(A) polymerase in E.coli HeLa cells and calf thymus respectively. Clones were only A fragment containing the open reading frame of the bovine obtained from the HeLa library. Although these clones clone was inserted into a T7 expression vector such that the encoded the correct amino acid sequences, they had an open initiating ATG codon was that encoding the first amino acid reading frame for a protein of only 43 kDa and lacked of the authentic protein (see Materials and methods). peptide 66 (see below). Therefore, a fragment from the Depending on the induction conditions, the synthesis of a coding region of these clones was used to screen an additional polypeptide of the expected size could be barely or not at cDNA library from calf muzzle epithelium. The largest all detected by SDS-PAGE of total cell lysates. After cDNA clone obtained in this screen was 2.5 kb in length induction overnight at 18°C, nonspecific poly(A) polymerase (Figure 2). It contained an open reading frame coding for activity, measured by the incorporation of radiolabeled ATP a protein of 739 amino acids with a molecular weight of into acid-precipitable material in the presence of a poly(A) GCGGTTGCGGGGGGGAAGTGACTGGGCGGTGCGGCGCAGGAGACGATGCCGTTTCCAGTTACAACACAGGGATCACAGCAAACACAGCCG 90 M P F P V T T O G S 0 0 T O P 15 CCACAGAAGCACTATGGCATTACTTCTCCCATCAGCTTAGCAGCCCCCAAGGAGACTGACTGCCTGCTCACACAGAAGCTGGTGGAGACT 180 P O K H Y G I T S P I S L A A P K E T D C L L T Q K L V E T 45 CTGAAGCCCTTCGGGGTTTTTGAAGAGGAAGAGGAACTGCAGCGCAGGATTTTTATTTTGGGAAAATTAAATAACCTGGTAAAAGAGTGG 270 L K P F G V F E E E E E L Q R R I F I L G K L N N L V K E W 75 ATACGAGAAATCAGTGAAAGCAAGAATCTTCCACAATCTGTAATTGAAAATGTTGGTGGGAAAATTTTTACATTTGGATCTTATAGATTA 360 I R E I S E S K N L P Q S V I E N V G G K I F T F G S Y R L 105 GGAGTACATACAAAAGGTGCTGATATTGATGCATTGTGTGTTGCACCAAGACATGTTGATCGAAGTGATTTTTTCACCTCATTCTATGAT 450 G V H T K G A D I D A L C V A P R H V D R S D F F T S F Y D 135 AAGTTGAAATTACAGGAAGAAGTAAAAGATTTAAGAGCTGTTGAAGAGGCATTTGTACCAGTTATCAAACTGTGTTTTGATGGGATAGAG 540 K L K L Q E E V K D L R A V E E A F V P V I K L C F D G I E 165 ATTGATATTTTGTTTGCAACATTAGCACTGCAGACTATTCCAGAAGACTTGGACTTAAGAGATGACAGTCTGCTTAAAAATTTAGATATA 630 I D I L F A R L A L Q T I P E D L D L R D D S L L K N L D I 195 AGATGTATAAGAAGTCTTAACGGTTGCAGGGTAACCGATGAAATTTTACATCTAGTACCAAACATTGACAACTTCAGGTTAACCCTGAGA 720 R C I R S L N G C R V T D E L H L V P N I D N F R L T L R 225 GCTATCAAACTGTGGGCCAAACGCCACAACATCTATTCCAATATATTAGGTTTCCTCGGTGGTGTTTCCTGGGCTATGCTAGTAGCAAGA 810 A I K L W A K R H N I Y S N I L G F L G G V S W A M L V A R 255 ACTTGCCAGCTTTATCCAMATGCAATAGCATCAACTCTTGTACATAAATTTTTCTTGGTATTTTCTAAATGGGAATGGCCAAATCCAGTC 900 T C Q L Y P N A I A S T L V H K F F L V F S K W E W P N P V 285 CTATTGAAACAGCCTGAAGAATGCAATCTTAATTTGCCTGTATGGGACCCAAGGGTAAACCCCAGTGATAGGTACCATCTTATGCCTATA 990 L L K Q P E E C N L N L P V W D P R V N P S D R Y H L M P I 315 ATTACACCAGCATACCCACAACAGAACTCCACGTACAATGTGTCCGTTTCAACACGGATGGTCATGGTTGAGGAGTTTAAACAAGGTCTT 1080 I T P A Y P Q Q N S T Y N V S V S T R M V M V E E F K Q G L 345 GCTATCACAGATGAAATTTTGCTGAGTAAGGCAGAGTGGTCCAAACTTTTTGAAGCTCCAAACTTCTTTCAAAAGTACCAGCATGATATT 1170 A I T D E I L L S K A E W S K L F E A P N F F Q K Y Q H D I 375 GTACTTCTAGCAAGTGCACCAACTGAAAAACAACGCCTAGAATGGGTGGGCTTGGTGGAATCAAAAATCCGAATCCTGGTTGGAAGTTTG 1260 V L L A S A P T E K O R L E W V G L V E S K I R I L V G S L 405 GAGAAGAATGAGTTTATGACACTGGCTCATGTGAATCCCCAGTCATTTCCAGCACCCAAAGAAAATCCCGACAAGGAAGAATTTCGCACT 1350 E K N E F M T L A H V N P Q S F P A P K E N P D K E E F R T 435 ATGTGGGTGATTGGGTTAGTGTTTAACAAAACAGAAAACTCTGAAAATCTCAGTGTTGATCTCACCTATGATATTCAGTCTTTCACAGAT 1440 M W V I G L V F N K T E N S E N L S V D L T Y D I Q S F T D 465 ACAGTTTATAGGCAAGCAATAAACAGCAAGATGTTTGAGGTGGACATGAAAATTGCTGCGATGCATGTAAAAAGAAAGCAACTCCATCAA
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