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J. Biol. Chem., Vol. 283, Issue 10, 6145-6153, March 7, 2008

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Ricin Inhibits Activation of the Unfolded Protein Response by Preventing Splicing of the HAC1 mRNA^*

From the Biotechnology Center for Agriculture and the Environment and the Department of Plant Biology and Pathology, School of Environmental and Biological Sciences, Rutgers University, New Brunswick, New Jersey 08901-8520

Ricin A chain (RTA) inhibits protein synthesis by removing a specific adenine from the highly conserved -sarcin/ricin loop in the large rRNA. Expression of RTA with its own signal sequence in yeast resulted in its translocation into the endoplasmic reticulum (ER) and subsequent glycosylation. Because RTA must unfold within the ER, it may be vulnerable to host defenses, such as the unfolded protein response (UPR). UPR was induced in cells expressing an active site mutant but not the wild type RTA, indicating that the active site of RTA played a role in perturbing the ER stress response. The inactive RTA without the signal sequence did not induce UPR, indicating that translocation into the ER was critical for induction of UPR. The wild type RTA inhibited activation of UPR not only due to ER stress induced by the protein itself but also by global effectors such as tunicamycin and dithiothreitol. Mature RTA without the signal sequence also inhibited UPR, providing evidence that inhibition of UPR occurred on the cytosolic face of the ER. RTA could not inhibit UPR when the spliced form of HAC1 mRNA was provided in trans, indicating that it had a direct effect on UPR upstream of HAC1-dependent transcriptional activation. Only the precursor form of HAC1 mRNA was detected in cells expressing RTA after exposure to ER stress, demonstrating that ricin inhibits activation of UPR by preventing HAC1 mRNA splicing. The RTA mutants that depurinated ribosomes but did not kill cells were not able to inhibit activation of UPR by tunicamycin, providing evidence that the inability to activate UPR in response to ER stress contributes to the cytotoxicity of ricin.

Received for publication, September 24, 2007 , and in revised form, December 14, 2007.

^* This work was supported in part by National Institutes of Health Grant AI072425 (to N. E. T.) and National Science Foundation Grant MCB0348299 (to N. E. T.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ Supported by a National Science Foundation Research Experience for Undergraduates grant.

² To whom correspondence should be addressed: Biotechnology Center, Foran Hall, SEBS, 59 Dudley Rd., New Brunswick, NJ 08901-8520. Tel.: 732-932-8165 (ext. 215); Fax: 732–932-6535; E-mail: tumer{at}aesop.rutgers.edu.

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