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J. Biol. Chem., Vol. 277, Issue 44, 41428-41437, November 1, 2002
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From the Pokeweed antiviral protein (PAP), a
single chain ribosome-inactivating protein (RIP) isolated from pokeweed
plants (Phytolacca americana), removes specific adenine and
guanine residues from the highly conserved,
Pokeweed Antiviral Protein Regulates the Stability of Its Own
mRNA by a Mechanism That Requires Depurination but Can Be Separated
from Depurination of the
-Sarcin/Ricin Loop of rRNA*
§,, and§¶
Biotechnology Center for Agriculture and the
Environment and the Department of Plant Biology and Pathology Cook
College, Rutgers University and the § Graduate Program in
Microbiology and Molecular Genetics, Rutgers University, New Brunswick,
New Jersey 08901-8520
-sarcin/ricin
loop in the large rRNA, resulting in inhibition of protein synthesis.
We recently demonstrated that PAP could also inhibit translation of
mRNAs and viral RNAs that are capped by binding to the cap
structure and depurinating the RNAs downstream of the cap. Cell growth
is inhibited when PAP cDNA is expressed in the yeast
Saccharomyces cerevisiae under the control of the
galactose-inducible GAL1 promoter. Here, we show that
overexpression of wild type PAP in yeast leads to a decrease in PAP
mRNA abundance. The decrease in mRNA levels is not observed
with an active site mutant, indicating that it is due to the
N-glycosidase activity of the protein. PAP expression had
no effect on steady state levels of mRNA from four different endogenous yeast genes examined, indicating specificity. We demonstrate that PAP can depurinate the rRNA in trans in a
translation-independent manner. When rRNA is depurinated and
translation is inhibited, the steady state levels of PAP mRNA
increase dramatically relative to the U3 snoRNA. Using a PAP variant
which depurinates rRNA, inhibits translation but does not destabilize
its mRNA, we demonstrate that PAP mRNA is destabilized after
its levels are up-regulated by a mechanism that occurs independently of
rRNA depurination and translation. We quantify the extent of rRNA
depurination in vivo using a novel primer extension assay
and show that the temporal pattern of rRNA depurination is similar to
the pattern of PAP mRNA destabilization, suggesting that they may
occur by a common mechanism. These results provide the first in
vivo evidence that a single chain RIP targets not only the large
rRNA but also its own mRNA. These findings have implications
for understanding the biological function of RIPs.
*
This work was supported by National Science Foundation Grant
MCB 9982498 (to N. E. T.).The costs of publication of this
article were defrayed in part by the
payment of page charges. The article must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
Copyright © 2002 by The American Society for Biochemistry and Molecular Biology, Inc.
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