Definition of an efficient synthetic poly(A) site.
We constructed and analyzed a synthetic poly(A) (SPA) site that was based on the highly efficient poly(A) signal of the rabbit beta-globin gene. By use of the SPA, we demonstrate that the minimum sequences required for efficient polyadenylation are the AATAAA sequence and a GT/T-rich sequence with the correct spacing of 22-23 nucleotides between them. When placed downstream of the poly(A) site of the human alpha 2-globin gene, the SPA is used exclusively. We predict that the SPA, with its more extensive GT/T-rich sequence, is a more efficient poly(A) site than alpha-globin. Also, we compared the use of the SPA when it is placed either in the exon 3 or intron 2 of the rabbit beta-globin gene. When in the exonic position, SPA is used 10-fold more than the regular poly(A) site of rabbit beta-globin. In contrast, when it is in the intronic location, no detectable use of SPA is observed; however, the deletion of the donor site of intron 2 reactivates the intronic positioned SPA. These results indicate that the splicing of intron 2 in the rabbit beta-globin gene occurs ahead of polyadenylation and have important implications for termination of transcription. Polyadenylation, although required for termination of transcription, is not sufficient; therefore, additional termination signals for RNA polymerase II must exist.
Levitt
N
N
Sir William Dunn School of Pathology, University of Oxford, UK.
Briggs
D
D
Gil
A
A
Proudfoot
N J
NJ
D000818:Animals; D001483:Base Sequence; D004247:DNA / Q000235:genetics; D005091:Exons; D005092:Exonucleases / Q000378:metabolism; D005914:Globins / Q000235:genetics*; D006801:Humans; D007438:Introns; D008969:Molecular Sequence Data; D009693:Nucleic Acid Hybridization; D011061:Poly A / Q000235:genetics*; D012326:RNA Splicing; D012333:RNA, Messenger / Q000235:genetics*; D011817:Rabbits; D015183:Restriction Mapping; D014158:Transcription, Genetic
D012333:RNA, Messenger; D011061:Poly A; D005914:Globins; D004247:DNA; D005092:Exonucleases