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Molecular and Cellular Biology, September 2005, p. 7675-7686, Vol. 25, No. 17
0270-7306/05/$08.00+0     doi:10.1128/MCB.25.17.7675-7686.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Eukaryotic Translational Coupling in UAAUG Stop-Start Codons for the Bicistronic RNA Translation of the Non-Long Terminal Repeat Retrotransposon SART1

Kenji K. Kojima,{dagger} Takumi Matsumoto, and Haruhiko Fujiwara*

Department of Integrated Biosciences, Graduate School of Frontier Sciences, University of Tokyo, Kashiwa-shi, Chiba 277-8562, Japan

Received 16 December 2004/ Returned for modification 19 January 2005/ Accepted 14 June 2005

Most eukaryotic cellular mRNAs are monocistronic; however, many retroviruses and long terminal repeat (LTR) retrotransposons encode multiple proteins on a single RNA transcript using ribosomal frameshifting. Non-long terminal repeat (non-LTR) retrotransposons are considered the ancestor of LTR retrotransposons and retroviruses, but their translational mechanism of bicistronic RNA remains unknown. We used a baculovirus expression system to produce a large amount of the bicistronic RNA of SART1, a non-LTR retrotransposon of the silkworm, and were able to detect the second open reading frame protein (ORF2) by Western blotting. The ORF2 protein was translated as an independent protein, not as an ORF1-ORF2 fusion protein. We revealed by mutagenesis that the UAAUG overlapping stop-start codon and the downstream RNA secondary structure are necessary for efficient ORF2 translation. Increasing the distance between the ORF1 stop codon and the ORF2 start codon decreased translation efficiency. These results are different from the eukaryotic translation reinitiation mechanism represented by the yeast GCN4 gene, in which the probability of reinitiation increases as the distance between the two ORFs increases. The translational mechanism of SART1 ORF2 is analogous to translational coupling observed in prokaryotes and viruses. Our results indicate that translational coupling is a general mechanism for bicistronic RNA translation.

* Corresponding author. Mailing address: Department of Integrated Biosciences, Graduate School of Frontier Sciences, University of Tokyo, Bioscience Building, 501, Kashiwa-shi, Chiba 277-8562, Japan. Phone: 81-4-7136-3659. Fax: 81-4-7136-3660. E-mail: haruh{at}k.u-tokyo.ac.jp.

{dagger} Present address: Bioinformatics Center, Institute for Chemical Research, Kyoto University, Uji, Kyoto 611-0011, Japan.

Molecular and Cellular Biology, September 2005, p. 7675-7686, Vol. 25, No. 17
0022-538X/05/$08.00+0     doi:10.1128/MCB.25.17.7675-7686.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.



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