Specific pre-cleavage and post-cleavage complexes involved in the formation of SV40 late mRNA 3' termini in vitro

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Updated: Aug 7, 2026

Analysis of RNA Processing Reactions Using Cell Free Systems: 3' End Cleavage of Pre-mRNA Substrates in vitro
09:16

Analysis of RNA Processing Reactions Using Cell Free Systems: 3' End Cleavage of Pre-mRNA Substrates in vitro

Published on: May 3, 2014

D Zarkower1, M Wickens

  • 1Department of Biochemistry, College of Agriculture and Life Sciences, University of Wisconsin-Madison 53706.

The EMBO Journal
|December 20, 1987
PubMed

Insights

Processing factors bind simian virus 40 (SV40) pre-mRNA, forming pre- and post-cleavage complexes. The conserved AAUAAA sequence is crucial for complex formation and RNA processing at the polyadenylation site.

Area of Science:

  • Molecular Biology
  • Virology
  • RNA Processing

Background:

  • Nuclear extracts contain processing factors that interact with simian virus 40 (SV40) RNA.
  • SV40 late pre-mRNA contains a polyadenylation [poly(A)] site crucial for processing.
  • The AAUAAA sequence is a conserved element involved in RNA polyadenylation.

Purpose of the Study:

  • To investigate the formation and requirements of complexes between nuclear processing factors and SV40 late pre-mRNA.
  • To determine the role of the AAUAAA sequence in pre-mRNA cleavage and complex formation.
  • To characterize the distinct complexes formed before and after pre-mRNA cleavage.

Main Methods:

  • Utilized crude nuclear extracts from HeLa cells.
  • Analyzed complex formation with SV40 late pre-mRNA spanning the poly(A) site.
  • Investigated the effect of mutations in the AAUAAA sequence and DNA oligonucleotide annealing.
  • Performed competition experiments to assess factor requirements.

Main Results:

  • A specific pre-cleavage complex forms on SV40 pre-mRNA, requiring the AAUAAA sequence.
  • Mutations in AAUAAA or annealing DNA oligonucleotides prevent pre-cleavage complex formation.
  • A distinct post-cleavage complex forms containing the 5' half-molecule after cleavage.
  • The 3' half-molecule and polyadenylated RNA are released after processing.
  • De novo post-cleavage complex formation requires AAUAAA and a nearby 3' terminus.

Conclusions:

  • The AAUAAA sequence is essential for both pre- and post-cleavage complex formation during SV40 RNA processing.
  • A component recognizing AAUAAA is critical for the assembly of these processing complexes.
  • Distinct molecular events and complexes characterize the cleavage and polyadenylation steps.

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