Identification of sequences in the herpes simplex virus thymidine kinase gene required for efficient processing and polyadenylation.

Abstract

The herpes simplex virus (HSV) type 1 thymidine kinase gene (tk) was resected from its 3' end with BAL 31 exonuclease. Two sets of plasmids were isolated that lacked information distal to the two copies of the hexanucleotide 5'-AATAAA-3' located at the 3' end of the HSV tk gene. The presence of a simian virus 40 origin of DNA replication in each plasmid facilitated analysis of patterns of transcription in transfected Cos-1 monkey cells. Transcription analyses were performed with an Si nuclease protection assay. Efficient processing and polyadenylation at the normal site still occurred when all sequences more than 44 or 46 base pairs (bp) downstream from the first AATAAA were removed (pTK311R/SVO10 and pTK209R/SVO10). Removal of an additional 7 bp (pTK312R/SVO10) decreased the amount of tk mRNA processed at that normal site, and tk mRNA polyadenylated at a cryptic site within pBR322 sequences began to appear. The normal processing and polyadenylation site was not used at all when an additional 12 bp was removed (pTK314R/SVO10); the small amount of tk mRNA produced was processed and polyadenylated at the cryptic pBR322 site. The region of the tk gene critical for efficient processing and polyadenylation of tk mRNA is located 20 to 38 bp downstream from the first AATAAA, distal to the polyadenylation site, and as RNA can form a stem-loop structure containing AAUAAA. Similar G+T-rich elements were located in DNA fragments which substitute efficiently for the HSV tk processing and polyadenylation signal and were not found in AATAAA-containing DNA fragments which substitute inefficiently for the HSV tk signal.The biosynthesis of eucaryotic mRNA requires a series of processing steps which convert the precursor heterogeneous nuclear RNA into functional mRNA (see reference 38 for a review). These processing steps include the addition of caps at the 5' end of the mature mRNA, the removal of intervening sequences by splicing, and the addition of polyadenylic acid at the 3' end of the mature mRNA. Although transcription by RNA polymerase II initiates at the cap site, evidence from several systems (17, 18,22,39) indicates that transcription extends beyond the site of polyadenylation and that endonucleolytic cleavage creates the site at which a poly(A) polymerase adds approximately 200 A residues.

A dissection of the cauliflower mosaic virus polyadenylation signal.

“…1). Two TG-rich regions, resembling the TG-rich sequence found downstream of the poly(A) addition site in vertebrate genes (Gil and Proudfoot 1984;Coles and Stacy 1985;Conway and Wickens 1985;Sadofsky et al 1985) and possibly in plant genes (Joshi 1987), are found 70 and 124 nucleotides upstream of the processing site. A third TG-rich region is located 32 nucleotides upstream of the processing site and consists of several tandem repeats of the sequence TTTGTA (Fig.…”

“…In addition, a less well-conserved T-rich or TG-rich element, usually situated imiPresent address: Agriculture Canada Research Station, Vancouver, B.C., V6T 1X2 Canada. mediately downstream of the poly(A) addition site, is important for both reactions in vivo and in vitro (Gil and Proudfoot 1984;Coles and Stacy 1985;Conway and Wickens 1985;Hart et al 1985;McLauchlan et al 1985;Sadofsky et al 1985;Sperry and Berget 1986). Both the AATAAA and the downstream element are required for formation of the specific cleavage and polyadenylation complexes (Skolnik-David et al 1987;Gilmartin and Nevins 1989).…”

Direct interaction of the U1 snRNP-A protein with the upstream efficiency element of the SV40 late polyadenylation signal.

“…Examination of many polyadenylation signals has shown that besides the conserved AAUAAA sequence, there are surrounding RNA sequences required for efficient utilization of the AAU-AAA as a polyadenylation signal. Downstream efficiency elements (DSEs) have been identified in many different viral systems (Gil andProudfoot 1984, 1987;McDevitt et al 1984McDevitt et al , 1986Sadofsky and Alwine 1984;Cole and Stacy 1985;Conway and Wickens 1985;Sadofsky et al 1985;Zhang and Cole 1987;Wilusz et al 1988;Wilusz and Shenk 1990) at distances from 5 to 60 nucleotides downstream of the poly(A) addition site. These 'Coiiesponding author. elements do not conform to a consensus sequence but are characterized as either U-rich or GU-rich.…”

A multicomponent complex is required for the AAUAAA-dependent cross-linking of a 64-kilodalton protein to polyadenylation substrates.

“…This cleavage-polyadenylation event is mediated through two cis-acting sequence elements: a highly conserved hexanucleotide, AAUAAA, located 5 to 30 bases upstream of the cleavage site (8,12,22,27,35,37), and a less conserved U-or GU-rich stretch located downstream of the cleavage site (6,7,10,17,19,20,28,30,31). The selective recognition of alternative polyadenylation sites may play a role in the posttranscriptional regulation of gene expression (reviewed in reference 14).…”