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Plant and Cell Physiology Advance Access originally published online on January 9, 2008
Plant and Cell Physiology 2008 49(3):314-323; 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 email: 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, Hokkaido University, Kita-ku, Sapporo, 060-8589 Japan
2Division of Life Science, Graduate School of Life Science, Hokkaido University, Kita-ku, Sapporo, 060-8589 Japan

*Corresponding author: E-mail, naito{at}abs.agr.hokudai.ac.jp; Fax, +81-11-706-4932.


  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 monosomes, whereas longer degradation intermediates were associated with multiple ribosomes. Immunoblot 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 ribosomes loaded on mRNA increased. These results show that AdoMet induces the stacking of ribosomes on CGS1 mRNA and that multiple mRNA degradation sites probably correspond to each stacked ribosome.

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

Abbreviations: AdoMet, S-adenosyl-L-methionine; CGS, cystathionine {gamma}-synthase; GST, glutathione S-transferase; LUC, firefly luciferase; NGD, no-go decay; WGE, wheat germ extract.

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

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