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. Articles by others on the same topic
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