Fine-structure analysis of the processing and polyadenylation region of the herpes simplex virus type 1 thymidine kinase gene by using linker scanning, internal deletion, and insertion mutations.

Abstract

Most eucaryotic mRNAs are polyadenylated. In higher eucaryotes, the sequence AATAAA is located 7 to 30 base pairs (bp) upstream from the site of processing and polyadenylation and is a critical part of the signal for processing and polyadenylation. Efficient cleavage and polyadenylation also require sequences downstream of polyadenylation sites. The herpes simplex virus type 1 thymidine kinase (tk) gene contains two copies of the AATAAA hexanucleotide and a GT box (18 of 19 consecutive residues are G or T) previously shown to be required for efficient processing and polyadenylation of tk mRNA (C. N. Cole and T. P. Stacy, Mol. Cell. Biol., 5:2104-2113). To define further the sequence requirements for efficient polyadenylation, we prepared linker scanning, internal deletion, and small insertion mutations in the polyadenylation region of the tk gene. These mutations were analyzed by S1 nuclease protection analysis of cytoplasmic RNA isolated from transfected Cos-1 monkey kidney cells. When the proximal AATAAA was deleted, no tk mRNA polyadenylated in the normal region was detected, whereas replacement of the second AATAAA with an XbaI linker had no effect on polyadenylation. When various portions of the GT box were replaced with linker, the amount of tk mRNA produced was reduced to 23 to 82% of the normal amount, but polyadenylation in the normal region was never abolished. Thus, no single portion of the GT box was absolutely required. In some cases, extended transcripts, polyadenylated at a cryptic site within pBR322, were detected. A spacing of 6 bp between AATAAA and the GT box was too short for efficient processing and polyadenylation. A spacing of 30 bp appeared to work almost as efficiently as did the wild-type spacing of 18 bp. Taken together, these results indicate that efficient polyadenylation requires both AATAAA and downstream GT-rich sequences. In addition, processing and polyadenylation are affected both qualitatively and quantitatively by sequences at polyadenylation sites and at more distant locations.