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RNA sequence containing hexanucleotide AAUAAA directs efficient mRNA polyadenylation in vitro.

J L Manley, H Yu, L Ryner

    Molecular and Cellular Biology
    |February 1, 1985
    PubMed
    Summary
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    A specific AAUAAA sequence near the 3' end of RNA directs polyadenylation. This sequence enables polyadenylic acid polymerase to add a poly-A tail, even when the 3' end is distant.

    Area of Science:

    • Molecular Biology
    • RNA Processing
    • Gene Expression

    Background:

    • Polyadenylation is a crucial post-transcriptional modification of eukaryotic messenger RNA (mRNA).
    • The precise sequence requirements and mechanisms governing polyadenylation in vitro are not fully elucidated.
    • Simian virus 40 (SV40) early pre-mRNA serves as a model system for studying RNA processing events.

    Purpose of the Study:

    • To investigate the role of specific nucleotide sequences in directing polyadenylation of SV40 early pre-mRNA.
    • To determine if polyadenylation can occur in a soluble HeLa whole-cell lysate.
    • To identify the minimal sequence requirements for in vitro polyadenylation.

    Main Methods:

    • Construction of rearranged and deleted DNA templates encoding SV40 early pre-mRNA.

    Related Experiment Videos

  • In vitro transcription of DNA templates to generate pre-mRNAs.
  • Incubation of synthesized RNAs with a soluble HeLa whole-cell lysate to assess polyadenylation.

    • Main Results:

    • Transferring a 237-bp fragment encoding the 3' end of SV40 early pre-mRNA to non-polyadenylated RNAs enabled efficient polyadenylation.
    • Deletion mutant analysis revealed that the AAUAAA sequence near the 3' end is essential for in vitro polyadenylation.
    • The AAUAAA sequence directed polyadenylation even when located up to 400 nucleotides upstream of the RNA 3' end.

    Conclusions:

    • A specific nucleotide sequence (AAUAAA) is required for directing polyadenylation of SV40 early pre-mRNA in HeLa cell lysates.
    • Polyadenylic acid polymerase activity in HeLa lysates can recognize the AAUAAA sequence and utilize the RNA 3' end as a primer for polyadenylation.
    • This process occurs in the absence of the nucleolytic cleavage typically observed in vivo, suggesting a distinct in vitro mechanism.