Incubate a labelled precursor messenger RNA containing an authentic -processing signal with a HeLa nuclear extract, then resolve the products by denaturing polyacrylamide gel electrophoresis. With ATP, the upstream cleavage product acquires a heterogeneous poly(A) tail. Replace ATP with an appropriate non-incorporable analogue, such as alpha,beta-methylene-ATP: a discrete product ending at the normal cleavage site still appears, but the extended tail does not. Thus cleavage can occur when polyadenylation is blocked; it is not cleavage brought about by synthesis of the tail.[ref]
Accurate cleavage and polyadenylation of exogenous RNA substrate by Claire L. Moore and Phillip A. Sharp. Abstract: cleavage without polyadenylation in the presence of alpha,beta-methylene-ATP
Conversely, supply an RNA substrate already ending at the normal cleavage site: addition of ATP and the appropriate poly(A) polymerase machinery can extend its free hydroxyl without any new endonucleolytic cut.[ref]
The AAUAAA sequence is required both for cleavage and for polyadenylation of simian virus 40 pre-mRNA in vitro by David Zarkower and others. Abstract: efficient polyadenylation of pre-cleaved RNA substrates in nuclear extract
Compare precursor, pre-cleaved substrate, no-extract and no-ATP controls, and map the product ends to distinguish correct processing from non-specific degradation. These are separable reactions, normally coordinated in one processing complex. Their separability does not imply that they use completely independent recognition factors.