ELAV proteins stabilize deadenylated intermediates in a novel in vitro mRNA deadenylation/degradation system

We have developed an in vitro mRNA stability system using HeLa cell cytopla smic S100 extracts and exogenous polyadenylated RNA substrates that reprodu ces regulated aspects of mRNA decay. The addition of cold poly(A) competito r RNA activated both a sequence-specific deadenylase activity in the extrac ts as well as a potent, ATP-dependent ribonucleolytic activity. The rates o f both deadenylation and degradation were up-regulated by the presence of a variety of AU-rich elements in the body of substrate RNAs. Competition ana lyses demonstrated that trans-acting factors were required for RNA destabil ization by AU-rich elements. The similar to 30-kD ELAV protein HuR specific ally bound to RNAs containing an AU-rich element derived from the TNF-alpha mRNA in the in vitro system. Interaction of HuR with AU-rich elements, how ever, was not associated with RNA destabilization. Interestingly, recombina nt ELAV proteins specifically stabilized deadenylated intermediates generat ed from the turnover of AU-rich element-containing substrate RNAs. These da ta suggest that mammalian ELAV proteins play a role in regulating mRNA stab ility by influencing the access of degradative enzymes to RNA substrates.