Components required for in vitro cleavage and polyadenylation of eukaryotic mRNA.
We have studied in vitro cleavage/polyadenylation of precursor RNA containing herpes simplex virus type 2 poly A site sequences and have analyzed four RNA/protein complexes which form during in vitro reactions. Two complexes, A and B, form extremely rapidly and are then progressively replaced by a third complex, C which is produced following cleavage and polyadenylation of precursor RNA. Substitution of ATP with cordycepin triphosphate prevents polyadenylation and the formation of complex C however a fourth complex, D results which contains cleaved RNA. A precursor RNA lacking GU-rich downstream sequences required for efficient cleavage/polyadenylation fails to form complex B and produces a markedly reduced amount of complex A. As these GU-rich sequences are required for efficient cleavage, this establishes a relationship between complex B formation and cleavage/polyadenylation of precursor RNA in vitro. The components required for in vitro RNA processing have been separated by fractionation of the nuclear extract on Q-Sepharose and Biorex 70 columns. A Q-Sepharose fraction forms complex B but does not process RNA. Addition of a Biorex 70 fraction restores cleavage activity at the poly A site but this fraction does not appear to contribute to complex formation. Moreover, in the absence of polyethylene glycol, precursor RNA is not cleaved and polyadenylated, however, complexes A and B readily form. Thus, while complex B is necessary for in vitro cleavage and polyadenylation, it may not contain all the components required for this processing.
D001483:Base Sequence; D002467:Cell Nucleus / Q000378:metabolism; D006367:HeLa Cells / Q000378:metabolism; D006801:Humans; D010957:Plasmids; D011061:Poly A / Q000235:genetics* / Q000378:metabolism; D011485:Protein Binding; D012322:RNA Precursors / Q000235:genetics*; D012333:RNA, Messenger / Q000235:genetics*; D018139:Simplexvirus / Q000235:genetics
D012322:RNA Precursors; D012333:RNA, Messenger; D011061:Poly A