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Research Papers
Department of Microbiology and Molecular Genetics, Markey Center for Molecular Genetics, University of Vermont, Burlington 05405.
Abstract
The endonucleolytic cleavage and polyadenylation of a pre-mRNA in mammalian cells requires two cis-acting elements, a highly conserved AAUAAA hexamer and an amorphous U- or GU-rich downstream element, that together constitute the "core" poly(A) site. The terminal redundancy of the HIV-1 pre-mRNA requires that the processing machinery disregard a core poly(A) site at the 5' end of the transcript, and efficiently utilize an identical signal that resides near the 3' end. Efficient processing at the 3' core poly(A) site, both in vivo and in vitro, has been shown to require sequences 76 nucleotides upstream of the AAUAAA hexamer. In this report we demonstrate that this HIV-1 upstream element interacts directly with the 160-kD subunit of CPSF (cleavage polyadenylation specificity factor), the factor responsible for the recognition of the AAUAAA hexamer. The presence of the upstream element in the context of the AAUAAA hexamer directs the stable binding of CPSF to the pre-mRNA and enhances the efficiency of poly(A) addition in reactions reconstituted with purified CPSF and recombinant poly(A) polymerase. Our results indicate that the dependence of HIV-1 3' processing on upstream sequences is a consequence of the suboptimal sequence context of the AAUAAA hexamer. We suggest that poly(A) site definition involves the recognition of multiple heterogeneous sequence elements in the context of the AAUAAA hexamer.
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