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Vol. 20, Issue 13, 3025-3032, July 1, 2009

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Analysis of Dom34 and Its Function in No-Go Decay

Dario O. Passos^*, Meenakshi K. Doma^*,, Christopher J. Shoemaker, Denise Muhlrad^*, Rachel Green, Jonathan Weissman, Julie Hollien^,||, and Roy Parker^*

*University of Arizona, Department of Molecular and Cellular Biology and Howard Hughes Medical Institute, Tucson, AZ 85721; Howard Hughes Medical Institute, University of California, San Francisco, San Francisco, CA 94158-2330; and Howard Hughes Medical Institute, Department of Molecular Biology and Genetics, Johns Hopkins University School of Medicine, Baltimore, MD 21205

Submitted January 12, 2009; Revised April 23, 2009; Accepted April 29, 2009
Monitoring Editor: Marvin Wickens

Eukaryotic mRNAs are subject to quality control mechanisms that degrade defective mRNAs. In yeast, mRNAs with stalls in translation elongation are targeted for endonucleolytic cleavage by No-Go decay (NGD). The cleavage triggered by No-Go decay is dependent on Dom34p and Hbs1p, and Dom34 has been proposed to be the endonuclease responsible for mRNA cleavage. We created several Dom34 mutants and examined their effects on NGD in yeast. We identified mutations in several loops of the Dom34 structure that affect NGD. In contrast, mutations inactivating the proposed nuclease domain do not affect NGD in vivo. Moreover, we observed that overexpression of the Rps30a protein, a high copy suppressor of dom34 cold sensitivity, can restore some mRNA cleavage in a dom34 strain. These results identify important functional regions of Dom34 and suggest that the proposed endonuclease activity of Dom34 is not required for mRNA cleavage in NGD. We also provide evidence that the process of NGD is conserved in insect cells. On the basis of these results and the process of translation termination, we suggest a multistep model for the process of NGD.

Present addresses: Division of Biology, California Institute of Technology, Pasadena, CA 91125;

^||Department of Biology, University of Utah, Salt Lake City, UT 84112.

Address correspondence to: Roy Parker (rrparker{at}email.arizona.edu)