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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