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PMID: 2538718 Published · ppublish English Journal Article Research Support, Non-U.S. Gov't

Poly(A) polymerase purified from HeLa cell nuclear extract is required for both cleavage and polyadenylation of pre-mRNA in vitro.

Molecular and cellular biology ·Vol. 9 ·No. 1 ·1989-01-00 ·Pages 193-203

Christofori G, Keller W

Abstract

We have partially purified a poly(A) polymerase (PAP) from HeLa cell nuclear extract which is involved in the 3'-end formation of polyadenylated mRNA. PAP had a molecular weight of approximately 50 to 60 kilodaltons. In the presence of manganese ions, PAP was able to polyadenylate RNA nonspecifically. However, in the presence of magnesium ions PAP required the addition of a cleavage and polyadenylation factor to specifically polyadenylate pre-mRNAs that contain an intact AAUAAA sequence and end at the poly(A) addition site (precleaved RNA substrates). The purified fraction containing PAP was also required in combination with a cleavage and polyadenylation factor and a cleavage factor for the correct cleavage at the poly(A) site of pre-mRNAs. Since the two activities of the PAP fractions, PAP and cleavage activity, could not be separated by extensive purification, we concluded that the two activities are contained in a single component, a PAP that is also required for the specific cleavage preceding the polyadenylation of pre-mRNA.

MeSH Terms
Cell Extracts Cells, Cultured Chemical Fractionation Chromatography HeLa Cells Humans Nucleotidyltransferases/metabolism Plasmids Poly A/genetics,metabolism Polynucleotide Adenylyltransferase/genetics,metabolism RNA Precursors/metabolism RNA Splicing RNA, Messenger/metabolism RNA, Nuclear/metabolism
Chemicals
Cell Extracts RNA Precursors RNA, Messenger RNA, Nuclear Poly A Nucleotidyltransferases Polynucleotide Adenylyltransferase
Authors & Affiliations
2 authors, click to expand affiliations / ORCID
Christofori G
Department of Cell Biology, University of Basel, Switzerland.
Keller W
References (54)
54 references, click to expand
  1. Electrophoretic separation of polyadenylation-specific complexes.
    Genes Dev. 1987 Sep;1(7):672-82 PMID: 3428596
  2. Sedimentation analysis of polyadenylation-specific complexes.
    Mol Cell Biol. 1988 Jan;8(1):226-33 PMID: 2961980
  3. Separation and characterization of a poly(A) polymerase and a cleavage/specificity factor required for pre-mRNA polyadenylation.
    Cell. 1988 Mar 11;52(5):731-42 PMID: 2830992
  4. Cleavage and polyadenylation of messenger RNA precursors in vitro occurs within large and specific 3' processing complexes.
    EMBO J. 1987 Dec 20;6(13):4159-68 PMID: 3127203
  5. Analysis of mRNA 3' end formation by modification interference: the only modifications which prevent processing lie in AAUAAA and the poly(A) site.
    EMBO J. 1987 Dec 20;6(13):4177-84 PMID: 3443104
  6. Specific pre-cleavage and post-cleavage complexes involved in the formation of SV40 late mRNA 3' termini in vitro.
    EMBO J. 1987 Dec 20;6(13):4185-92 PMID: 2832155
  7. A functionally redundant downstream sequence in SV40 late pre-mRNA is required for mRNA 3'-end formation and for assembly of a precleavage complex in vitro.
    J Biol Chem. 1988 Apr 25;263(12):5780-8 PMID: 2833517
  8. Structural and functional characterization of mouse U7 small nuclear RNA active in 3' processing of histone pre-mRNA.
    Mol Cell Biol. 1988 Apr;8(4):1518-24 PMID: 3380087
  9. Mutations in poly(A) site downstream elements affect in vitro cleavage activity.
    Mol Cell Biol. 1988 Apr;8(4):1839-41 PMID: 2837659
  10. Multiple factors are required for specific RNA cleavage at a poly(A) addition site.
    Genes Dev. 1988 May;2(5):578-87 PMID: 2838381
  11. Multiple factors are required for poly(A) addition to a mRNA 3' end.
    Genes Dev. 1988 May;2(5):588-97 PMID: 3384332
  12. Assembly of a polyadenylation-specific 25S ribonucleoprotein complex in vitro.
    Mol Cell Biol. 1988 May;8(5):2052-62 PMID: 2898729
  13. Specific contacts between mammalian U7 snRNA and histone precursor RNA are indispensable for the in vitro 3' RNA processing reaction.
    EMBO J. 1988 Mar;7(3):801-8 PMID: 3396543
  14. 3' cleavage and polyadenylation of mRNA precursors in vitro requires a poly(A) polymerase, a cleavage factor, and a snRNP.
    Cell. 1988 Sep 9;54(6):875-89 PMID: 2842067
  15. Two proteins crosslinked to RNA containing the adenovirus L3 poly(A) site require the AAUAAA sequence for binding.
    EMBO J. 1988 Oct;7(10):3159-69 PMID: 3181133
  16. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding.
    Anal Biochem. 1976 May 7;72:248-54 PMID: 942051
  17. 3' non-coding region sequences in eukaryotic messenger RNA.
    Nature. 1976 Sep 16;263(5574):211-4 PMID: 822353
  18. Isolation and partial characterization of the poly(A) polymerases from HeLa cells infected with vaccinia virus.
    J Biol Chem. 1977 Oct 10;252(19):6939-47 PMID: 893453
  19. The sequence 5'-AAUAAA-3'forms parts of the recognition site for polyadenylation of late SV40 mRNAs.
    Cell. 1981 Apr;24(1):251-60 PMID: 6113054
  20. Simple, efficient in vitro synthesis of capped RNA useful for direct expression of cloned eukaryotic genes.
    Nucleic Acids Res. 1982 Oct 25;10(20):6353-62 PMID: 6294601
  21. Accurate transcription initiation by RNA polymerase II in a soluble extract from isolated mammalian nuclei.
    Nucleic Acids Res. 1983 Mar 11;11(5):1475-89 PMID: 6828386
  22. Inhibition of RNA cleavage but not polyadenylation by a point mutation in mRNA 3' consensus sequence AAUAAA.
    Nature. 1983 Oct 13-19;305(5935):600-5 PMID: 6194440
  23. Splicing of in vitro synthesized messenger RNA precursors in HeLa cell extracts.
    Cell. 1983 Nov;35(1):89-99 PMID: 6194902
  24. Alpha-thalassaemia caused by a polyadenylation signal mutation.
    Nature. 1983 Nov 24-30;306(5941):398-400 PMID: 6646217
  25. Site-specific polyadenylation in a cell-free reaction.
    Cell. 1984 Mar;36(3):581-91 PMID: 6230155
  26. Requirement of a downstream sequence for generation of a poly(A) addition site.
    Cell. 1984 Jul;37(3):993-9 PMID: 6744418
  27. The 5' terminus of the RNA moiety of U1 small nuclear ribonucleoprotein particles is required for the splicing of messenger RNA precursors.
    Cell. 1984 Aug;38(1):299-307 PMID: 6235919
  28. Efficient in vitro synthesis of biologically active RNA and RNA hybridization probes from plasmids containing a bacteriophage SP6 promoter.
    Nucleic Acids Res. 1984 Sep 25;12(18):7035-56 PMID: 6091052
  29. Role of the conserved AAUAAA sequence: four AAUAAA point mutants prevent messenger RNA 3' end formation.
    Science. 1984 Nov 30;226(4678):1045-51 PMID: 6208611
  30. A sequence downstream of AAUAAA is required for rabbit beta-globin mRNA 3'-end formation.
    Nature. 1984 Nov 29-Dec 5;312(5993):473-4 PMID: 6095108
  31. RNA sequence containing hexanucleotide AAUAAA directs efficient mRNA polyadenylation in vitro.
    Mol Cell Biol. 1985 Feb;5(2):373-9 PMID: 2579321
  32. Transcription termination and 3' processing: the end is in site!
    Cell. 1985 Jun;41(2):349-59 PMID: 2580642
  33. The consensus sequence YGTGTTYY located downstream from the AATAAA signal is required for efficient formation of mRNA 3' termini.
    Nucleic Acids Res. 1985 Feb 25;13(4):1347-68 PMID: 2987822
  34. A sequence downstream of A-A-U-A-A-A is required for formation of simian virus 40 late mRNA 3' termini in frog oocytes.
    Proc Natl Acad Sci U S A. 1985 Jun;82(12):3949-53 PMID: 2987956
  35. Accurate cleavage and polyadenylation of exogenous RNA substrate.
    Cell. 1985 Jul;41(3):845-55 PMID: 2408761
  36. Stepwise assembly of a pre-mRNA splicing complex requires U-snRNPs and specific intron sequences.
    Cell. 1985 Aug;42(1):355-67 PMID: 3160483
  37. The conserved CAAGAAAGA spacer sequence is an essential element for the formation of 3' termini of the sea urchin H3 histone mRNA by RNA processing.
    EMBO J. 1985 Feb;4(2):481-9 PMID: 2410259
  38. Poly(A) site cleavage in a HeLa nuclear extract is dependent on downstream sequences.
    Cell. 1985 Dec;43(3 Pt 2):677-83 PMID: 2866847
  39. Purification of a protein required for the splicing of pre-mRNA and its separation from the lariat debranching enzyme.
    EMBO J. 1985 Dec 16;4(13A):3571-81 PMID: 4092689
  40. A small nuclear ribonucleoprotein associates with the AAUAAA polyadenylation signal in vitro.
    Cell. 1986 May 23;45(4):581-91 PMID: 2423249
  41. Identification of a sequence element on the 3' side of AAUAAA which is necessary for simian virus 40 late mRNA 3'-end processing.
    Mol Cell Biol. 1985 Oct;5(10):2713-9 PMID: 3016512
  42. U1, U2, and U4/U6 small nuclear ribonucleoproteins are required for in vitro splicing but not polyadenylation.
    Cell. 1986 Aug 29;46(5):691-6 PMID: 2427201
  43. Analysis of RNA cleavage at the adenovirus-2 L3 polyadenylation site.
    EMBO J. 1986 Aug;5(8):1929-38 PMID: 3019671
  44. The AAUAAA sequence is required both for cleavage and for polyadenylation of simian virus 40 pre-mRNA in vitro.
    Mol Cell Biol. 1986 Jul;6(7):2317-23 PMID: 3023928
  45. In vitro cleavage of the simian virus 40 early polyadenylation site adjacent to a required downstream TG sequence.
    Mol Cell Biol. 1986 Dec;6(12):4734-41 PMID: 3025668
  46. The role of small nuclear ribonucleoprotein particles in pre-mRNA splicing.
    Nature. 1987 Feb 19-25;325(6106):673-8 PMID: 2950324
  47. Requirements for accurate and efficient mRNA 3' end cleavage and polyadenylation of a simian virus 40 early pre-RNA in vitro.
    Mol Cell Biol. 1987 Jan;7(1):495-503 PMID: 3031477
  48. Formation of mRNA 3' termini: stability and dissociation of a complex involving the AAUAAA sequence.
    EMBO J. 1987 Jan;6(1):177-86 PMID: 2438129
  49. Products of in vitro cleavage and polyadenylation of simian virus 40 late pre-mRNAs.
    Mol Cell Biol. 1987 Apr;7(4):1518-29 PMID: 3037325
  50. Identification of a complex associated with processing and polyadenylation in vitro of herpes simplex virus type 1 thymidine kinase precursor RNA.
    Mol Cell Biol. 1987 Sep;7(9):3277-86 PMID: 2823124
  51. Fractionation of HeLa cell nuclear extracts reveals minor small nuclear ribonucleoprotein particles.
    Proc Natl Acad Sci U S A. 1987 Dec;84(23):8408-12 PMID: 2960976
  52. Identification of the human U7 snRNP as one of several factors involved in the 3' end maturation of histone premessenger RNA's.
    Science. 1987 Dec 18;238(4834):1682-7 PMID: 2825355
  53. Separation of multiple components of HeLa cell nuclear extracts required for pre-messenger RNA splicing.
    J Biol Chem. 1987 Dec 25;262(36):17630-40 PMID: 2961739
  54. A 64 kd nuclear protein binds to RNA segments that include the AAUAAA polyadenylation motif.
    Cell. 1988 Jan 29;52(2):221-8 PMID: 2830023
Article Info
Journal
Molecular and cellular biology
Abbr.
Mol Cell Biol
ISSN
0270-7306
DOI
10.1128/mcb.9.1.193-203.1989
Published
1989-01-00
Pages
193-203
Language
English
Region
United States
NLM ID
8109087
PMCID
PMC362161
Subset
IM
External Links
PubMed source DOI: 10.1128/mcb.9.1.193-203.1989 PMC Full Text View journal details
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