Definition of an efficient synthetic poly(A) site.

Abstract

We constructed and analyzed a synthetic poly(A) (SPA) site that was based on the highly efficient poly(A) signal of the rabbit I~-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 a2-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 c~-globin. Also, we compared the use of the SPA when it is placed either in the exon 3 or intron 2 of the rabbit 13-globin gene. When in the exonic position, SPA is used 10-fold more than the regular poly(A) site of rabbit 13-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 13-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.

A dissection of the cauliflower mosaic virus polyadenylation signal.

“…In plants, the analysis of the pea ribulose-r5-biphosphate carboxylase small-subunit gene suggested the involvement of several upstream and downstream elements (Hunt and MacDonald 1989). In vertebrate systems, it has been shown that an AATAAA and a downstream element are the only necessary requirements for efficient processing and that the polyadenylation site is usually at an A residue (Kessler et al 1986;Levitt et al 1989). However, upstream elements in addition to the downstream element have been described for the SV40 late poly (A) signal (Carswell and Alwine 1989), for the adenovirus late transcription unit (DeZasso and Imperiale 1989), and for the hepatitis B virus (Russnak and Ganem 1990).…”

Ending the message: poly(A) signals then and now

“…Interestingly, this is achieved by the dominant role of the 59SS and its recognition by U1snRNP, which act to block adjacent PAS recognition. Again, this phenomenon was first described in gene-specific studies (Levitt et al 1989;Zhao et al 1999) but has now achieved genome-wide status (Kaida et al 2010). Early examples of such regulation were found in mammalian viruses, which often need to maximize their gene expression output by selective use of PAS.…”

A shortened adeno-associated virus expression cassette for CFTR gene transfer to cystic fibrosis airway epithelia

“…Poly(A) signals range from 600 to Ͼ1,000 bp. However, previous studies have shown that the required consensus sequence, AATAAAX 22 GT x ͞T x , in a 49-bp sequence produces protein expression equivalent to that of a full-length poly(A) signal in HeLa cells (26). We compared this 49-bp SPA with the 624-bp hGH poly(A) signal present in pCMV5-␤-gal.…”