Poly(A) site cleavage in a HeLa nuclear extract is dependent on downstream sequences.

Efficient utilization of the early SV40 poly(A) site in vivo requires sequences between 5 bp and 18 bp downstream of the cleavage site. We have used a HeLa nuclear extract to examine the sequence requirements for in vitro cleavage. DNA segments containing the SV40 poly(A) site were cloned into an SP6 vector. SP6 RNAs, accurately cleaved and polyadenylated, were detected by primer extension. Cleavage was enhanced by the presence of a cap on the primary transcript, and was inhibited by the addition of 10 microM 7meGpppG. In close agreement with the in vivo results, efficient processing at the poly(A) site in vitro required the specific downstream sequences in the SP6 RNA transcript. These experiments indicate that the sequence in the RNA precursor downstream of the cleavage site, shown to be important for efficient processing in vivo, is recognized in vitro.