Nucleic Acids Research, Vol 27, Issue 2 446-454, Copyright © 1999 by Oxford University Press
BI Klasens, M Thiesen, A Virtanen and B Berkhout
The 5' and 3' ends of HIV-1 transcripts are identical in sequence. This
repeat region (R) folds a stem-loop structure that is termed the poly(A)
hairpin because it contains polyadenylation or poly(A) signals: the AAUAAA
hexamer motif, the cleavage site and part of the GU-rich downstream
element. Obviously, HIV-1 gene expression necessitates differential
regulation of the two poly(A) sites. Previous transfection experiments
indicated that the wild-type poly(A) hairpin is slightly inhibitory to the
process of polyadenylation, and further stabilization of the hairpin
inhibited polyadenylation completely. In this study, we tested wild-type
and mutant transcripts with poly(A) hairpin structures of differing
thermodynamic stabilities for the in vitro binding of polyadenylation
factors. Mutant transcripts with a destabilized hairpin efficiently bound
the polyadenylation factors, which were provided either as purified
proteins or as nuclear extract. The RNA mutant with a stabilized hairpin
did not form this 'poly(A) complex'. Additional mutations that repair the
stability of this hairpin restored the binding capacity. Thus, an inverse
correlation was measured between the stability of the poly(A) hairpin and
its ability to interact with polyadenylation factors. The wild-type HIV-1
transcript bound the polyadenylation factors suboptimally, but full
activity was obtained in the presence of the USE enhancer element that is
uniquely present upstream of the 3' poly(A) site. We also found that
sequences of the HIV-1 leader, which are uniquely present downstream of the
5' poly(A) site, inhibit formation of the poly(A) complex. This inhibition
could not be ascribed to a specific leader sequence, as we measured a
gradual loss of complex formation with increasing leader length. We will
discuss the regulatory role of RNA structure and the repressive effect of
leader sequences in the context of differential HIV-1 polyadenylation.
ARTICLES
The ability of the HIV-1 AAUAAA signal to bind polyadenylation factors is controlled by local RNA structure
Department of Human Retrovirology, Academic Medical Center, University of Amsterdam, Meibergdreef 15,1105 AZ Amsterdam, The Netherlands.
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