Components required for in vitro cleavage and polyadenylation of eukaryotic mRNA

McLauchlan, J., Moore, C.L., Simpson, S. and Clements, J.B. (1988) Components required for in vitro cleavage and polyadenylation of eukaryotic mRNA. Nucleic Acids Research, 16(12), pp. 5323-5344. (doi:10.1093/nar/16.12.5323)

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We have studied in vitro cleavage/polyadenylation of precursor RNA containing herpes simplex virus type 2 poly A site sequences and have analysed 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.

Item Type:Articles
Glasgow Author(s) Enlighten ID:McLauchlan, Professor John
Authors: McLauchlan, J., Moore, C.L., Simpson, S., and Clements, J.B.
College/School:College of Medical Veterinary and Life Sciences > Institute of Infection Immunity and Inflammation
Journal Name:Nucleic Acids Research
Publisher:Oxford University Press
ISSN (Online):1362-4962
Copyright Holders:Copyright © 1988 IRL Press Limited
First Published:First published in Nucleic Acids Research 16(12):5323-5344
Publisher Policy:Reproduced in accordance with the copyright policy of the publisher

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