= Solution
Incubate a labelled precursor <messenger RNA> containing an authentic $3'$-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 \b[cleavage can occur when <polyadenylation> is blocked]; it is not cleavage brought about by synthesis of the tail.<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]{ref}
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 $3'$ hydroxyl without any new endonucleolytic cut.<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]{ref} Compare precursor, pre-cleaved substrate, no-extract and no-<ATP> controls, and map the product ends to distinguish correct processing from non-specific degradation. \b[These are separable reactions, normally coordinated in one processing complex.] Their separability does not imply that they use completely independent recognition factors.
Back to article page
