Nucleic Acids Research, Vol 26, Issue 8 1870-1876, Copyright © 1998 by Oxford University Press
BI Klasens, AT Das and B Berkhout
The presence of a polyadenylation signal in the repeat (R) region of the
HIV-1 genome, which is located at both the 5' and 3' ends of the viral
transcripts, requires differential regulation of polyadenylation. The HIV-1
poly(A) site can fold in a stable stem-loop structure that is
well-conserved among different human and simian immunodeficiency viruses.
In this study, we tested the effect of this hairpin on polyadenylation by
introducing mutations that either stabilize or destabilize the RNA
structure. The HIV-1 sequences were inserted into the pSV2CAT reporter
plasmid upstream of the SV40 early poly(A) site. These constructs were
transfected into COS cells and transcripts were analyzed for the usage of
the HIV-1 versus SV40 poly(A) site. The wild- type HIV-1 poly(A) site was
used efficiently in this context and destabilization of the poly(A) hairpin
did not affect the polyadenylation efficiency. In contrast, further
stabilization of the hairpin severely inhibited HIV-1 polyadenylation.
Additional mutations that repair the thermodynamic stability of this mutant
hairpin restored the polyadenylation activity. These results indicate that
the mechanism of polyadenylation can be repressed by stable RNA structure
encompassing the poly(A) signal. Experiments performed at reduced
temperatures also suggest an inverse correlation between the stability of
the RNA structure and the efficiency of polyadenylation.
ARTICLES
Inhibition of polyadenylation by stable RNA secondary structure
Department of Human Retrovirology, Academic Medical Center, University of Amsterdam, Meibergdreef 15, 1105 AZ Amsterdam, PO Box 22700, 1100 DE Amsterdam, The Netherlands.
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