RNA sequence containing hexanucleotide AAUAAA directs efficient mRNA polyadenylation in vitro

To determine whether a specific nucleotide sequence is required to direct polyadenylation of a simian virus 40 early pre-mRNA in a soluble HeLa whole-cell lysate, we constructed a series of rearranged and deleted DNA templates, transcribed them in vitro, and determined whether the resultant RNAs could be polyadenylated when incubated in whole-cell lysate. When a 237-base-pair DNA fragment encoding the 3' end of the simian virus 40 early pre-mRNA was transferred to recombinant plasmids encoding RNAs that were not substrates for polyadenylation, the resultant RNAs could now be polyadenylated efficiently. In one case, the chimeric RNA was polyadenylated even more efficiently than was the original simian virus 40 early transcript. Analysis of the RNAs produced from the deletion mutant templates revealed that only RNAs containing at least one copy of the AAUAAA sequence situated near the 3' end and implicated in 3'-end formation and polyadenylation in vivo could be polyadenylated in vitro. Surprisingly, this sequence directed polyadenylation of pre-mRNAs not only when near the RNA 3' end, i.e., 50 nucleotides or less away, but also when the 3' end was situated over 400 nucleotides downstream. Thus, our results show that a polyadenylic acid polymerase activity in HeLa lysates can recognize a specific nucleotide sequence in pre-mRNA and then, in the absence of the nucleolytic cleavage that presumably occurs in vivo, locate the RNA 3' end and use it as a primer for polyadenylic acid synthesis.

[1]  J. Sodroski,et al.  Repetitive structure in the long-terminal-repeat element of a type II human T-cell leukemia virus. , 1984, Proceedings of the National Academy of Sciences of the United States of America.

[2]  T. Yasunaga,et al.  Bovine leukemia virus: unique structural features of its long terminal repeats and its evolutionary relationship to human T-cell leukemia virus. , 1984, Proceedings of the National Academy of Sciences of the United States of America.

[3]  S. Berget.  Are U4 small nuclear ribonucleoproteins involved in polyadenylation? , 1984, Nature.

[4]  P. Sharp,et al.  Site-specific polyadenylation in a cell-free reaction , 1984, Cell.

[5]  M. Perricaudet,et al.  Polyadenylic Acid Addition Sites in the Adenovirus Type 2 Major Late Transcription Unit , 1984, Journal of virology.

[6]  S. Goodbourn,et al.  α-Thalassaemia caused by a polyadenylation signal mutation , 1983, Nature.

[7]  Craig Montell,et al.  Inhibition of RNA cleavage but not polyadenylation by a point mutation in mRNA 3′ consensus sequence AAUAAA , 1983, Nature.

[8]  H. Boedtker,et al.  Multiple 3' ends of the chicken pro α2(1) collagen gene , 1983 .

[9]  J. Manley.  Accurate and specific polyadenylation of mRNA precursors in a soluble whole-cell lysate , 1983, Cell.

[10]  M. Yoshida,et al.  Human adult T-cell leukemia virus: complete nucleotide sequence of the provirus genome integrated in leukemia cell DNA. , 1983, Proceedings of the National Academy of Sciences of the United States of America.

[11]  J. Manley.  Analysis of the expression of genes encoding animal mRNA by in vitro techniques. , 1983, Progress in nucleic acid research and molecular biology.

[12]  James E. Darnell,et al.  Variety in the level of gene control in eukaryotic cells , 1982, Nature.

[13]  J. Darnell,et al.  The primary transcription unit of the mouse β-Major globin gene , 1981, Cell.

[14]  J. Manley,et al.  DNA sequence required for initiation of transcription in vitro from the major late promoter of adenovirus 2. , 1981, Proceedings of the National Academy of Sciences of the United States of America.

[15]  M. Perricaudet,et al.  Structure of two adenovirus type 12 transforming polypeptides and their evolutionary implications , 1980, Nature.

[16]  P. Sharp,et al.  DNA-dependent transcription of adenovirus genes in a soluble whole-cell extract. , 1980, Proceedings of the National Academy of Sciences of the United States of America.

[17]  R. Yuan,et al.  DNA translocation by the restriction enzyme from E. coli K , 1980, Cell.

[18]  C Benoist,et al.  The ovalbumin gene-sequence of putative control regions , 1980, Nucleic Acids Res..

[19]  D. Botstein,et al.  Advanced bacterial genetics , 1980 .

[20]  W. Gilbert,et al.  Sequencing end-labeled DNA with base-specific chemical cleavages. , 1980, Methods in enzymology.

[21]  J. Steitz,et al.  Antibodies to small nuclear RNAs complexed with proteins are produced by patients with systemic lupus erythematosus. , 1979, Proceedings of the National Academy of Sciences of the United States of America.

[22]  S. Weissman,et al.  Simian virus 40 early mRNA's. I. Genomic localization of 3' and 5' termini and two major splices in mRNA from transformed and lytically infected cells , 1979, Journal of virology.

[23]  J. Nevins,et al.  Steps in the processing of Ad2 mRNA: Poly(A)+ Nuclear sequences are conserved and poly(A) addition precedes splicing , 1978, Cell.

[24]  S. Weissman,et al.  The genome of simian virus 40. , 1978, Science.

[25]  S. Adhya,et al.  Control of transcription termination. , 1978, Annual review of biochemistry.

[26]  G. Carmichael,et al.  Analysis of single- and double-stranded nucleic acids on polyacrylamide and agarose gels by using glyoxal and acridine orange. , 1977, Proceedings of the National Academy of Sciences of the United States of America.

[27]  N. Proudfoot,et al.  3′ Non-coding region sequences in eukaryotic messenger RNA , 1976, Nature.