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Molecular and Cellular Biology, September 2004, p. 7902-7913, Vol. 24, No. 18
0270-7306/04/$08.00+0     DOI: 10.1128/MCB.24.18.7902-7913.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Essential Motifs in the 3' Untranslated Region Required for Retrotransposition and the Precise Start of Reverse Transcription in Non-Long-Terminal-Repeat Retrotransposon SART1

Mizuko Osanai, Hidekazu Takahashi, Kenji K. Kojima, Mitsuhiro Hamada, and Haruhiko Fujiwara^*

Department of Integrated Biosciences, Graduate School of Frontier Sciences, University of Tokyo, Kashiwa, Chiba, Japan

Received 14 October 2003/ Returned for modification 14 January 2004/ Accepted 2 June 2004

Non-long-terminal-repeat (non-LTR) retrotransposons amplify their copies by reverse transcribing mRNA from the 3' end, but the initial processes of reverse transcription are still unclear. We have shown that a telomere-specific non-LTR retrotransposon of the silkworm, SART1, requires the 3' untranslated region (3' UTR) for retrotransposition. With an in vivo retrotransposition assay, we identified several novel motifs within the 3' UTR involved in precise and efficient reverse transcription. Of 461 nucleotides (nt) of the 3' UTR, the central region, from nt 163 to nt 295, was essential for SART1 retrotransposition. Of five putative stem-loops formed in RNA for the SART1 3' UTR, the second stem-loop (nt 159 to 221) is included in this region. Loss of the 3' region (nt 296 to 461) in the 3' UTR and the poly(A) tract resulted in decreased and inaccurate reverse transcription, which starts mostly from several telomeric repeat-like GGUU sequences just downstream of the second stem-loop. These results suggest that short telomeric repeat-like sequences in the 3' UTR anneal to the bottom strand of (TTAGG)_n repeats. We also demonstrated that the mRNA for green fluorescent protein (GFP) could be retrotransposed into telomeric repeats when the GFP coding region is fused with the SART1 3' UTR and SART1 open reading frame proteins are supplied in trans.

Present address: Department of Molecular Biology and Genetics, Johns Hopkins University School of Medicine, Baltimore, MD 21205.

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