Restoration of both structure and function to a defective poly(A) site by in vitro selection.

The Journal of Biological Chemistry
B R GraveleyG M Gilmartin

Abstract

Efficient cleavage and polyadenylation at the human immunodeficiency virus type-1 (HIV-1) poly(A) site requires an upstream 3'-processing enhancer to overcome the suboptimal sequence context of the AAUAAA hexamer. The HIV-1 3'-processing enhancer functions to stabilize the association of the pre-mRNA with cleavage and polyadenylation specificity factor (CPSF), the factor responsible for recognition of the AAUAAA hexamer. Intriguingly, in the absence of the 3'-processing enhancer, CPSF binding and polyadenylation efficiency could be restored to near wild-type levels upon replacement of the 14-nucleotide region immediately 5' of the HIV-1 AAUAAA hexamer (the B segment) by the analogous sequences from the efficient adenovirus L3 poly(A) site. To further investigate the contributions of RNA sequence and structure to poly(A) site recognition, we have used an in vitro selection system to identify B segment sequences that enhance the polyadenylation efficiency of a pre-cleaved RNA lacking a 3'-processing enhancer. The final RNA selection pool was composed of two predominant classes of RNAs. Nuclease probing revealed that the selected sequences restored an RNA conformation that facilitates recognition of the AAUAAA hexamer by CPSF. The...Continue Reading

References

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Citations

Jun 17, 1999·FEMS Microbiology Reviews·E Wahle, U Rüegsegger
Nov 8, 2011·DNA and Cell Biology·Monica Lopes-MarquesLuisa Azevedo
Jun 6, 1998·Nucleic Acids Research·B I KlasensB Berkhout
Dec 18, 2001·The Journal of Biological Chemistry·Jean-Christophe PaillartRoland Marquet

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