Cleavage and polyadenylation of messenger RNA precursors in vitro occurs within large and specific 3' processing complexes

Abstract

We have investigated the assembly of complexes associated with in vitro cleavage and polyadenylation of synthetic pre-mRNAs by native gel electrophoresis. Incubation of SP6-generated pre-mRNA containing the adenovirus L3 polyadenylation site in HeLa cell nuclear extract results in the rapid assembly of specific complexes. Formation of these complexes precedes the appearance of cleaved intermediates and polyadenylated products and is dependent on an intact polyadenylation signal within the pre-mRNA. The specific complexes do not form on RNAs with point mutations in the AAUAAA sequence upstream of the L3 polyadenylation site. Furthermore, such mutant RNAs cannot compete for factors involved in the assembly of specific complexes on wild-type pre-mRNA. Upon complex formation a 67-nucleotide region of the L3 pre-mRNA is protected from RNase T1 digestion. This region contains both the upstream AAUAAA signal and the GU-rich downstream sequences. Cleavage and polyadenylation occur within the specific complexes and the processed RNA is subsequently released. We propose that the assembly of specific complexes represents an essential step during pre-mRNA 3' end formation in vitro.