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Trimeric structure and flexibility of the L1ORF1 protein in human L1 retrotransposition

Abstract

The LINE-1 (L1) retrotransposon emerges as a major source of human interindividual genetic variation, with important implications for evolution and disease. L1 retrotransposition is poorly understood at the molecular level, and the mechanistic details and evolutionary origin of the L1-encoded L1ORF1 protein (L1ORF1p) are particularly obscure. Here three crystal structures of trimeric L1ORF1p and NMR solution structures of individual domains reveal a sophisticated and highly structured, yet remarkably flexible, RNA-packaging protein. It trimerizes via an N-terminal, ion-containing coiled coil that serves as scaffold for the flexible attachment of the central RRM and the C-terminal CTD domains. The structures explain the specificity for single-stranded RNA substrates, and a mutational analysis indicates that the precise control of domain flexibility is critical for retrotransposition. Although the evolutionary origin of L1ORF1p remains unclear, our data reveal previously undetected structural and functional parallels to viral proteins.

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Figure 1: Domain structure of human L1ORF1p.
Figure 2: Crystal structure and domain organization of the human L1ORF1p trimer.
Figure 3: Flexibility of the L1ORF1p trimer.
Figure 4: Nucleic acid–binding properties of the L1ORF1p trimer.
Figure 5: Mutational analysis of L1ORF1p.
Figure 6: Structural parallels to viral proteins.

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References

  1. Goodier, J.L. & Kazazian, H.H. Jr. Retrotransposons revisited: the restraint and rehabilitation of parasites. Cell 135, 23–35 (2008).

    Article  CAS  PubMed  Google Scholar 

  2. Han, J.S. & Boeke, J.D. LINE-1 retrotransposons: modulators of quantity and quality of mammalian gene expression? Bioessays 27, 775–784 (2005).

    Article  CAS  PubMed  Google Scholar 

  3. Moran, J.V. & Gilbert, N. Mammalian LINE-1 retrotransposons and related elements. in Mobile DNA II Vol. 2 (eds. Craig, N.L., Craigie, R., Gellert, M. & Lambowitz, A.M.) 836–869 (ASM Press, 2002).

    Chapter  Google Scholar 

  4. Lander, E.S. et al. Initial sequencing and analysis of the human genome. Nature 409, 860–921 (2001).

    Article  CAS  PubMed  Google Scholar 

  5. Beck, C.R. et al. LINE-1 retrotransposition activity in human genomes. Cell 141, 1159–1170 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Ewing, A.D. & Kazazian, H.H. Jr. High-throughput sequencing reveals extensive variation in human-specific L1 content in individual human genomes. Genome Res. 20, 1262–1270 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Huang, C.R. et al. Mobile interspersed repeats are major structural variants in the human genome. Cell 141, 1171–1182 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Iskow, R.C. et al. Natural mutagenesis of human genomes by endogenous retrotransposons. Cell 141, 1253–1261 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Coufal, N.G. et al. L1 retrotransposition in human neural progenitor cells. Nature 460, 1127–1131 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Eickbush, T.H. & Malik, H.S. Origin and evolution of retrotransposons. in Mobile DNA II Vol. 2 (eds. Craig, N.L., Craigie, R., Gellert, M. & Lambowitz, A.M.) 1111–1144 (ASM Press, 2002).

    Chapter  Google Scholar 

  11. Cost, G.J., Feng, Q., Jacquier, A. & Boeke, J.D. Human L1 element target-primed reverse transcription in vitro. EMBO J. 21, 5899–5910 (2002).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Luan, D.D., Korman, M.H., Jakubczak, J.L. & Eickbush, T.H. Reverse transcription of R2Bm RNA is primed by a nick at the chromosomal target site: a mechanism for non-LTR retrotransposition. Cell 72, 595–605 (1993).

    Article  CAS  PubMed  Google Scholar 

  13. Khazina, E. & Weichenrieder, O. Non-LTR retrotransposons encode noncanonical RRM domains in their first open reading frame. Proc. Natl. Acad. Sci. USA 106, 731–736 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Moran, J.V. et al. High frequency retrotransposition in cultured mammalian cells. Cell 87, 917–927 (1996).

    Article  CAS  PubMed  Google Scholar 

  15. Hohjoh, H. & Singer, M.F. Cytoplasmic ribonucleoprotein complexes containing human LINE-1 protein and RNA. EMBO J. 15, 630–639 (1996).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Kulpa, D.A. & Moran, J.V. Ribonucleoprotein particle formation is necessary but not sufficient for LINE-1 retrotransposition. Hum. Mol. Genet. 14, 3237–3248 (2005).

    Article  CAS  PubMed  Google Scholar 

  17. Martin, S.L. Ribonucleoprotein particles with LINE-1 RNA in mouse embryonal carcinoma cells. Mol. Cell. Biol. 11, 4804–4807 (1991).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Goodier, J.L., Zhang, L., Vetter, M.R. & Kazazian, H.H. Jr. LINE-1 ORF1 protein localizes in stress granules with other RNA-binding proteins, including components of RNA interference RNA-induced silencing complex. Mol. Cell. Biol. 27, 6469–6483 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Soper, S.F. et al. Mouse maelstrom, a component of nuage, is essential for spermatogenesis and transposon repression in meiosis. Dev. Cell 15, 285–297 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Hohjoh, H. & Singer, M.F. Sequence-specific single-strand RNA binding protein encoded by the human LINE-1 retrotransposon. EMBO J. 16, 6034–6043 (1997).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Kolosha, V.O. & Martin, S.L. In vitro properties of the first ORF protein from mouse LINE-1 support its role in ribonucleoprotein particle formation during retrotransposition. Proc. Natl. Acad. Sci. USA 94, 10155–10160 (1997).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Martin, S.L. & Bushman, F.D. Nucleic acid chaperone activity of the ORF1 protein from the mouse LINE-1 retrotransposon. Mol. Cell. Biol. 21, 467–475 (2001).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Wei, W. et al. Human L1 retrotransposition: cis preference versus trans complementation. Mol. Cell. Biol. 21, 1429–1439 (2001).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Basame, S. et al. Spatial assembly and RNA binding stoichiometry of a LINE-1 protein essential for retrotransposition. J. Mol. Biol. 357, 351–357 (2006).

    Article  CAS  PubMed  Google Scholar 

  25. Martin, S.L., Branciforte, D., Keller, D. & Bain, D.L. Trimeric structure for an essential protein in L1 retrotransposition. Proc. Natl. Acad. Sci. USA 100, 13815–13820 (2003).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Januszyk, K. et al. Identification and solution structure of a highly conserved C-terminal domain within ORF1p required for tetrotransposition of long interspersed nuclear element-1. J. Biol. Chem. 282, 24893–24904 (2007).

    Article  CAS  PubMed  Google Scholar 

  27. Chou, K.C. Prediction of tight turns and their types in proteins. Anal. Biochem. 286, 1–16 (2000).

    Article  CAS  PubMed  Google Scholar 

  28. Hartmann, M.D. et al. A coiled-coil motif that sequesters ions to the hydrophobic core. Proc. Natl. Acad. Sci. USA 106, 16950–16955 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. Martin, S.L. et al. LINE-1 retrotransposition requires the nucleic acid chaperone activity of the ORF1 protein. J. Mol. Biol. 348, 549–561 (2005).

    Article  CAS  PubMed  Google Scholar 

  30. Martin, S.L. et al. A single amino acid substitution in ORF1 dramatically decreases L1 retrotransposition and provides insight into nucleic acid chaperone activity. Nucleic Acids Res. 36, 5845–5854 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. Evans, J.D., Peddigari, S., Chaurasiya, K.R., Williams, M.C. & Martin, S.L. Paired mutations abolish and restore the balanced annealing and melting activities of ORF1p that are required for LINE-1 retrotransposition. Nucleic Acids Res. doi:10.1093/nar/gkr171 (26 March 2011).

  32. Albertini, A.A., Schoehn, G., Weissenhorn, W. & Ruigrok, R.W. Structural aspects of rabies virus replication. Cell. Mol. Life Sci. 65, 282–294 (2008).

    Article  CAS  PubMed  Google Scholar 

  33. Raymond, D.D., Piper, M.E., Gerrard, S.R. & Smith, J.L. Structure of the Rift Valley fever virus nucleocapsid protein reveals another architecture for RNA encapsidation. Proc. Natl. Acad. Sci. USA 107, 11769–11774 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. Tawar, R.G. et al. Crystal structure of a nucleocapsid-like nucleoprotein-RNA complex of respiratory syncytial virus. Science 326, 1279–1283 (2009).

    Article  CAS  PubMed  Google Scholar 

  35. Ye, Q., Krug, R.M. & Tao, Y.J. The mechanism by which influenza A virus nucleoprotein forms oligomers and binds RNA. Nature 444, 1078–1082 (2006).

    Article  CAS  PubMed  Google Scholar 

  36. Cost, G.J. & Boeke, J.D. Targeting of human retrotransposon integration is directed by the specificity of the L1 endonuclease for regions of unusual DNA structure. Biochemistry 37, 18081–18093 (1998).

    Article  CAS  PubMed  Google Scholar 

  37. Repanas, K. et al. Determinants for DNA target structure selectivity of the human LINE-1 retrotransposon endonuclease. Nucleic Acids Res. 35, 4914–4926 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  38. Kapitonov, V.V. & Jurka, J. The esterase and PHD domains in CR1-like non-LTR retrotransposons. Mol. Biol. Evol. 20, 38–46 (2003).

    Article  CAS  PubMed  Google Scholar 

  39. Kammerer, R.A. et al. A conserved trimerization motif controls the topology of short coiled-coils. Proc. Natl. Acad. Sci. USA 102, 13891–13896 (2005).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  40. Kobe, B., Center, R.J., Kemp, B.E. & Poumbourios, P. Crystal structure of human T cell leukemia virus type 1 gp21 ectodomain crystallized as a maltose-binding protein chimera reveals structural evolution of retroviral transmembrane proteins. Proc. Natl. Acad. Sci. USA 96, 4319–4324 (1999).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  41. Weissenhorn, W., Dessen, A., Harrison, S.C., Skehel, J.J. & Wiley, D.C. Atomic structure of the ectodomain from HIV-1 gp41. Nature 387, 426–430 (1997).

    Article  CAS  PubMed  Google Scholar 

  42. Chen, J., Skehel, J.J. & Wiley, D.C. N- and C-terminal residues combine in the fusion-pH influenza hemagglutinin HA(2) subunit to form an N cap that terminates the triple-stranded coiled coil. Proc. Natl. Acad. Sci. USA 96, 8967–8972 (1999).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  43. Müller, M., Weigand, J.E., Weichenrieder, O. & Suess, B. Thermodynamic characterization of an engineered tetracycline-binding riboswitch. Nucleic Acids Res. 34, 2607–2617 (2006).

    Article  PubMed  PubMed Central  Google Scholar 

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Acknowledgements

We thank the staff at the Swiss Light Source (Villigen, Switzerland) for assistance during data collection, M.D. Hartmann for assistance with structure analysis and T. Holder for the movie script. Furthermore, we thank M. Fauser for an introduction to eukaryotic cell culture. We are grateful to E. Izaurralde for continued support and discussion. E.K. and O.W. were supported by a personal VIDI grant (NWO-CW 700.54.427) to O.W. from the Dutch National Science Organization (NWO).

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E.K. is the leading author, designed, performed and analyzed experiments, established cell culture, determined the crystal structures and wrote the manuscript. V.T. and M.C. recorded and analyzed NMR spectra, determined and analyzed NMR structures, and contributed to the manuscript. R.B. established cell culture, and performed and analyzed experiments. S.S. analyzed data and contributed to the manuscript. O.W. designed research, analyzed experiments and wrote the manuscript.

Corresponding author

Correspondence to Oliver Weichenrieder.

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Supplementary Text and Figures

Supplementary Figures 1–5 and Supplementary Methods (PDF 9351 kb)

Supplementary Movie 1

Flexibility and domain movements in the L1ORF1p trimer. While rotating once around the pseudo–three-fold axis of the trimer, the movie smoothly interpolates between the structural conformations observed in the three crystal forms (cfI, cfII and cfIII). Morphing transitions are from cfI via cfII to cfII and directly back to cfI. This is repeated once. The coiled coil is colored in gray, the RRM domains are in red and the CTD domains are in blue. Interdomain linkers are in lime and the central chloride ions are shown as yellow spheres. (MOV 12554 kb)

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Khazina, E., Truffault, V., Büttner, R. et al. Trimeric structure and flexibility of the L1ORF1 protein in human L1 retrotransposition. Nat Struct Mol Biol 18, 1006–1014 (2011). https://doi.org/10.1038/nsmb.2097

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