An in vitro system using HeLa cytoplasmic extracts that reproduces regulated mRNA stability

The pathways and machinery involved in the regulated turnover of mRNAs in m ammalian cells are largely unknown. We have developed an in vitro system us ing HeLa cytoplasmic S100 extracts and exogenous polyadenylated RNA substra tes that faithfully reproduces in vivo aspects of regulated mRNA turnover. RNA substrates for use in the system that contain a poly(A) tail precisely at their 3' end can be readily prepared using a ligation-polymerase chain r eaction approach. The system also uses standard cytoplasmic S100 extracts t hat are activated through the sequestration of poly(A)-binding proteins by the addition of cold poly(A) RNA. On incubation in the system, the poly(A) tail is removed from RNA substrates by a sequence-specific deadenylase acti vity and the body of the transcript is ultimately degraded in the system wi th no apparent intermediates by an ATP-dependent ribonulceolytic activity. AU-rich destability elements can regulate the rates of both deadenylation a nd degradation in the system. This in vitro system, therefore, should allow the elucidation of pathways of mRNA turnover, identification of the cellul ar factors involved, and insights into the mechanisms that regulate the hal f-life of a mRNA. (C) 1999 Academic Press.