THE 64-KILODALTON SUBUNIT OF THE CSTF POLYADENYLATION FACTOR BINDS TOPRE-MESSENGER-RNAS DOWNSTREAM OF THE CLEAVAGE SITE AND INFLUENCES CLEAVAGE SITE LOCATION

The CstF polyadenylation factor is a multisubunit complex required for efficient cleavage and polyadenylation of pre-mRNAs. Using an RNase H -mediated mapping technique, we show that the 64-kDa subunit of CstF c an be photo cross-linked to pre-mRNAs at U-rich regions located downst ream of the cleavage site of the simian virus 40 late and adenovirus L 3 pre-mRNAs. This positional specificity of cross-linking is a consequ ence of CstF interaction with the polyadenylation complex, since the 6 4-kDa protein by itself is cross-linked at multiple positions on a pre -mRNA template. During polyadenylation, four consecutive U residues ca n substitute for the native downstream U-rich sequence on the simian v irus 40 pre-mRNA, mediating efficient 64-kDa protein cross-linking at the downstream position. Furthermore, the position of the U stretch no t only enables the 64-kDa polypeptide to be cross-linked ts the pre-mR NA but also influences the site of cleavage. A search of the GenBank d atabase revealed that a substantial portion of mammalian polyadenylati on sites carried four or more consecutive U residues positioned so tha t they should function as sites for interaction with the 64-kDa protei n downstream of the cleavage site. Our results indicate that the polya denylation machinery physically spans the cleavage site, directing cle avage factors to a position located between the upstream AAUAAA motif, where the cleavage and polyadenylation specificity factor is thought to interact, and the downstream U-rich binding site for the 64-kDa sub unit of CstF.