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. 1988 May;8(5):2052–2062. doi: 10.1128/mcb.8.5.2052

Assembly of a polyadenylation-specific 25S ribonucleoprotein complex in vitro.

J E Stefano 1, D E Adams 1
PMCID: PMC363384  PMID: 2898729

Abstract

Extracts from HeLa cell nuclei assemble RNAs containing the adenovirus type 2 L3 polyadenylation site into a number of rapidly sedimenting heterodisperse complexes. Briefly treating reaction mixtures prior to sedimentation with heparin reveals a core 25S assembly formed with substrate RNA but not an inactive RNA containing a U----C mutation in the AAUAAA hexanucleotide sequence. The requirements for assembly of this heparin-stable core complex parallel those for cleavage and polyadenylation in vitro, including a functional hexanucleotide, ATP, and a uridylate-rich tract downstream of the cleavage site. The AAUAAA and a downstream U-rich element are resistant in the assembly to attack by RNase H. The poly(A) site between the two protected elements is accessible, but is attacked more slowly than in naked RNA, suggesting that a specific factor or secondary structure is located nearby. The presence of a factor bound to the AAUAAA in the complex is independently demonstrated by immunoprecipitation of a specific T1 oligonucleotide containing the element from the 25S fraction. Precipitation of this fragment from reaction mixtures is blocked by the U----C mutation. However, neither ATP nor the downstream sequence element is required for binding of this factor in the nuclear extract, suggesting that recognition of the AAUAAA is an initial event in complex assembly.

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