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A highly active synthetic mammalian retrotransposon

Abstract

LINE-1 (L1) elements are retrotransposons that comprise large fractions of mammalian genomes1. Transcription through L1 open reading frames is inefficient owing to an elongation defect2, inhibiting the robust expression of L1 RNA and proteins, the substrate and enzyme(s) for retrotransposition3,4,5. This elongation defect probably controls L1 transposition frequency in mammalian cells. Here we report bypassing this transcriptional defect by synthesizing the open reading frames of L1 from synthetic oligonucleotides, altering 24% of the nucleic acid sequence without changing the amino acid sequence. Such resynthesis led to greatly enhanced steady-state L1 RNA and protein levels. Remarkably, when the synthetic open reading frames were substituted for the wild-type open reading frames in an established retrotransposition assay4, transposition levels increased more than 200-fold. This indicates that there are probably no large, rigidly conserved cis-acting nucleic acid sequences required for retrotransposition within L1 coding regions. These synthetic retrotransposons are also the most highly active L1 elements known so far and have potential as practical tools for manipulating mammalian genomes.

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Figure 1: Synthesis and expression of synthetic mouse ORF2.
Figure 2: Retrotransposition of synthetic mL1.
Figure 3: Synthetic mouse L1 uses the standard retrotransposition mechanism.
Figure 4: High-frequency retrotransposition in mouse cells: total RNA analysis of smL1 expression.

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Acknowledgements

We thank Boeke laboratory members, especially Y. Aizawa, for helpful discussions and critical reading of the manuscript. We thank A. Yetil for assistance. This work was supported by the NIH (J.D.B.) and the Medical Scientist Training Program (J.S.H.).

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Correspondence to Jef D. Boeke.

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Supplementary information

Supplementary Legends

Legends for Supplementary Tables S1, S2 and Supplementary Figure S1 (DOC 30 kb)

Supplementary Tables

Supplementary Table S1:Oligonucleotides used; Supplementary Table S2: Codons used to synthesize smORF1 and smORF2. (PDF 17 kb)

Supplementary Figure S1

Alignment of native mouse L1 with synthetic mouse L1. (PDF 123 kb)

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Han, J., Boeke, J. A highly active synthetic mammalian retrotransposon. Nature 429, 314–318 (2004). https://doi.org/10.1038/nature02535

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