Designed and validated minimal synthetic polyadenylation signal (SPA) based on rabbit β-globin gene, demonstrating that only two elements are required for efficient polyadenylation: AAUAAA hexanucleotide and GT/T-rich sequence with 22-23 nucleotide spacing. SPA with extensive GT/T-rich sequence proved more efficient than natural α-globin signal and was used 10-fold more than native β-globin polyA when placed in exonic position, establishing design principles for minimal functional polyA signals.
Definition of an efficient synthetic poly(A) site
Parts used
Key findings
Abstract
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.