Anin VitroSystem Using HeLa Cytoplasmic Extracts That Reproduces Regulated mRNA Stability

Abstract

The pathways and machinery involved in the regulated turnover of mRNAs in mammalian cells are largely unknown. We have developed anin vitrosystem using HeLa cytoplasmic S100 extracts and exogenous polyadenylated RNA substrates that faithfully reproducesin vivoaspects 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 reaction approach. The system also uses standard cytoplasmic S100 extracts that 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 activity and the body of the transcript is ultimately degraded in the system with no apparent intermediates by an ATP-dependent ribonulceolytic activity. AU-rich destability elements can regulate the rates of both deadenylation and degradation in the system. Thisin vitrosystem, therefore, should allow the elucidation of pathways of mRNA turnover, identification of the cellular factors involved, and insights into the mechanisms that regulate the half-life of a mRNA.