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. These results indicate that both the sequence and structural context of the AAUAAA hexamer contribute to poly(A) site recognition by CPSF.
Highlights
The 3Ј-ends of all eukaryotic mRNAs are generated by endonucleolytic cleavage and, with the exception of those encoding histones, subsequently polyadenylated
The precise features of the cleavage and polyadenylation specificity factor (CPSF)-RNA interaction in the context of large RNA substrates will certainly be quite complex in vivo, the general themes revealed by in vitro analysis of smaller RNA substrates may provide an insight into the nature of poly(A) site recognition
We believe that the reconstituted poly(A) addition reaction, consisting of a precleaved RNA, purified human CPSF, and recombinant bovine PAP, represents a valid functional assay with which to explore the parameters governing poly(A) site recognition by CPSF
Summary
The 3Ј-ends of all eukaryotic mRNAs are generated by endonucleolytic cleavage and, with the exception of those encoding histones, subsequently polyadenylated. To examine the impact of RNA sequence and structure upon recognition of the AAUAAA hexamer by CPSF, we have used in vitro selection to identify B segment sequences capable of restoring efficient poly(A) addition to a pre-cleaved RNA containing the ⌬use mutation.
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