Multiple factors are required for specific RNA cleavage at a poly(A) addition site.

Abstract

An SP6 RNA containing the adenovirus 5 L3 poly(A) site is processed efficiently in a HeLa cell nuclear extract to generate correct 3' termini. Accurate 3' processing has also been demonstrated for the adenovirus E2A and SV40 early poly(A) sites, although these are processed less efficiently than the L3 site. Efficient cleavage at the poly(A) site requires the presence of a 5'-cap structure, as well as the RNA sequence motifs previously shown to be necessary for 3' processing in vivo, suggesting the presence and action of the appropriate factors in the nuclear extract. Fractionation of the nuclear extract has revealed a requirement for at least two distinct factors for cleavage at the L3 poly(A) site. One of these factors appears to possess an RNA component due to its sensitivity to micrococcal nuclease. The activity of this fraction is also sensitive to alpha-Sm monoclonal antibody, indicating the presence of an snRNP essential for the cleavage reaction. Additional factors are required for the subsequent polyadenylation reaction, indicating the involvement of a multicomponent complex in the processing of an RNA at the poly(A) site.