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Plant and Cell Physiology Advance Access published online on January 9, 2008
Plant and Cell Physiology, doi:10.1093/pcp/pcn005
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© The Author 2008. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For Permissions, please e-mail: journals.permissions@oxfordjournals.org

Ribosome Stacking Defines CGS1 mRNA Degradation Sites during Nascent Peptide-Mediated Translation Arrest

Yuhi Haraguchi1, Yoshitomo Kadokura2, Mari Nakamoto1,3, Hitoshi Onouchi1 and Satoshi Naito1,2,*

1Division of Applied Bioscience, Graduate School of Agriculture; and 2Division of Life Science, Graduate School of Life Science, Hokkaido University, Kita-ku, Sapporo 060-8589, Japan.

*Corresponding author:, Satoshi Naito, Graduate School of Life Science, Hokkaido University, Agriculture Bldg W501, Kita-9, Nishi-9, Kita-ku, Sapporo 060-8589, Japan, tel: +81-11-706-2800, fax: +91-11-706-4932, email: naito{at}abs.agr.hokudai.ac.jp


  Abstract

Expression of the Arabidopsis CGS1 gene that codes for cystathionine {gamma}-synthase is feedback-regulated at the step of mRNA degradation in response to S-adenosyl-L-methionine (AdoMet). This regulation occurs during translation and involves AdoMet-induced temporal translation arrest prior to the mRNA degradation. Here, we have identified multiple intermediates of CGS1 mRNA degradation with different 5’ ends that are separated by approximately 30 nucleotides. Longer intermediates were found to be produced as the number of ribosomes loaded on mRNA was increased. Sucrose density gradient centrifugation experiments showed that the shortest mRNA degradation intermediate was associated with monosome, whereas longer degradation intermediates were associated with multiple ribosomes. Immuno-blot analyses revealed a ladder of premature polypeptides whose molecular weights corresponded to products of ribosomes in a stalled stack. An increase in smaller premature polypeptides was observed as the number of ribosome loaded on mRNA increased. These results show that AdoMet induces the stacking of ribosomes on CGS1 mRNA and that multiple mRNA degradation sites likely correspond to each stacked ribosome.

Keywords: S-Adenosyl-L-methionine - Arabidopsis thaliana - Feedback regulation - Methionine biosynthesis - mRNA stability - MTO1 region

3Present address: Olympus Corp., Life Science Group, Hachioji 192-8512, Japan.

(Received December 9, 2007; Accepted January 6, 2008)
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