Abstract
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, alpha-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.
Highlights
Pokeweed antiviral protein (PAP),1 a single chain ribosomeinactivating protein (RIP) isolated from the leaves of pokeweed plants (Phytolacca americana), removes specific adenine and guanine residues from the highly conserved, ␣-sarcin/ricin (S/R) loop in the large rRNA [1,2,3]
PAP Inhibits the Growth of S. cerevisiae—To determine whether PAP affects the stability of its own mRNA in vivo, cDNAs encoding the wild type PAP or nontoxic variants were placed under the regulation of the GAL1 promoter and expressed in the yeast S. cerevisiae
PAP Has a Specific Effect on the Stability of Its Own mRNA in Yeast—Because our previous results had indicated that PAP can inhibit translation by depurinating capped RNAs [3], to determine whether translation inhibition correlated with the activity of PAP on mRNAs in vivo, we examined the abundance of PAP mRNA and cellular mRNAs in yeast expressing the wild type and mutant forms of PAP
Summary
Media and Growth Conditions—S. cerevisiae strain W303 (MATa ade trp ura leu 112 his can1-100) was used for all of the assays. 2 g of total yeast RNA from cells expressing PAP was hybridized with 106 cpm of reverse primer (5Ј-AGCGGATGGTGCTTCGCGGCAATG3Ј). This depurination primer was end-labeled by T4 kinase (Invitrogen) in the presence of [␥-32P]ATP, and it hybridized 73 nt 3Ј of the depurination site. Further studies requiring more accurate quantification of depurination employed the use of a second primer serving as an internal control For these analyses, either 1.25 g of total yeast RNA isolated from yeast expressing PAP, PAPE176V, or PAPL71R, as described above, or 1.0 g of rRNA isolated from ribosomes was hybridized to two different reverse primers. This amount was titrated previously to achieve 75% translation inhibition (data not shown)
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