AN EPISOMAL EXPRESSION VECTOR SYSTEM FOR MONITORING SEQUENCE-SPECIFICEFFECTS ON MESSENGER-RNA STABILITY IN HUMAN CELL-LINES

A plasmid expression system has been developed which allows sequence-s pecific effects on mRNA degradation rates to be determined. This syste m uses stable, nonintegrating vectors that provide consistent levels o f mRNA expression without the position effects common to integrating v ectors. cDNAs encoding putative instability elements may be subcloned into the 5' untranslated region (5'UTR), the coding region, or the pro ximal 3'UTR of a beta-globin cDNA reporter. The effects of these seque nces on mRNA stability may then be determined by actinomycin time cour se analyses of the fusion mRNAs and recombinant beta-globin mRNA in hu man cell lines. To demonstrate the utility of the vector system we fus ed an 820-bp fragment of the cDNA encoding the proximal 3'UTR of human 3-hydroxy-3-methylglutaryl coenzyme A (HMG CoA) reductase to the 3'UT R of the beta-globin reporter and introduced the vector into the human hepatocarcinoma cell line, HepG2. The fusion mRNA was degraded at a r ate 2- to 2.5-fold greater than that of beta-globin alone, at a rate s imilar to that reported for HMG CoA reductase mRNA in normal rat liver . Similar to a number of other relatively unstable mRNAs, the rate of fusion mRNA degradation was greatly decreased by treatment with cycloh eximide. (C) 1995 Academic Press, Inc.