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Cis-preferential LINE-1 reverse transcriptase activity in ribonucleoprotein particles

Nature Structural & Molecular Biology volume 13, pages 655–660 (2006)Cite this article

Abstract

LINE-1 retrotransposons (L1s) constitute ∼17% of human DNA, and their activity continues to affect genome evolution. Retrotransposition-competent human L1s encode two proteins required for their mobility (ORF1p and ORF2p); however, biochemical activities associated with ORF2p have been difficult to detect in cells. Here, we show for the first time the colocalization of L1 RNA, ORF1p and ORF2p to a putative ribonucleoprotein retrotransposition intermediate. We further demonstrate that ORF2p preferentially uses its encoding RNA as a template for reverse transcription. Thus, our data provide the first biochemical evidence supporting the cis-preferential action of the L1 reverse transcriptase.

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Figure 1: LEAP.
Figure 2: Schematic of sequenced LEAP and M-MLV RT-PCR products.
Figure 3: Effects of LEAP primer sequence on RT initiation.
Figure 4: Trans-LEAP.

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Acknowledgements

We thank members of the University of Michigan Sequencing Core for help with sequencing and S. King and A. Telesnitsky for help with quantitative PCR experiments. We also thank M. Imperiale, D. Friedman, J.L. Garcia-Perez, H. Chong and A. Hulme for comments and for critically reading the manuscript. The work was supported in part by a grant from the US National Institutes of Health (GM60518). D.A.K. was supported in part by a US National Institutes of Health training grant (GM07544). The University of Michigan Cancer Center helped defray some of the DNA-sequencing costs.

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Authors and Affiliations

  1. Department of Human Genetics 1241 E. Catherine St., University of Michigan Medical School, Ann Arbor, 48109-0618, Michigan, USA

    Deanna A Kulpa & John V Moran

  2. Department of Internal Medicine, 1241 E. Catherine St., University of Michigan Medical School, Ann Arbor, 48109-0618, Michigan, USA

    John V Moran

Authors
  1. Deanna A Kulpa
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Contributions

D.A.K. is the leading author. She contributed to the concept, designed and performed the experiments, analyzed the data and wrote the manuscript. J.V.M. is the senior author. He contributed to the concept, analyzed the data, revised the manuscript and provided financial support.

Corresponding authors

Correspondence to Deanna A Kulpa or John V Moran.

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The authors declare no competing financial interests.

Supplementary information

Supplementary Fig. 1

LEAP assay biophysical characterization (PDF 106 kb)

Supplementary Fig. 2

Effect of template-primer competition on LEAP activity (PDF 78 kb)

Supplementary Table 1

Sequences of oligonucleotide primers (PDF 18 kb)

Supplementary Methods (PDF 18 kb)

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Kulpa, D., Moran, J. Cis-preferential LINE-1 reverse transcriptase activity in ribonucleoprotein particles. Nat Struct Mol Biol 13, 655–660 (2006). https://doi.org/10.1038/nsmb1107

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