Template and target-site recognition by human LINE-1 in retrotransposition

Thawani, Akanksha; Ariza, Alfredo Jose Florez; Nogales, Eva; Collins, Kathleen

doi:10.1038/s41586-023-06933-5

Title: Template and target-site recognition by human LINE-1 in retrotransposition

Journal Article · 13 December 2023 · Nature (London)

DOI: https://doi.org/10.1038/s41586-023-06933-5 · OSTI ID:2470973

^[1];

^[2]; Collins, Kathleen ^[1]

California Institute for Quantitative Biosciences (QB3), Berkeley, CA (United States); University of California, Berkeley, CA (United States)
California Institute for Quantitative Biosciences (QB3), Berkeley, CA (United States); University of California, Berkeley, CA (United States); Howard Hughes Medical Institute, Chevy Chase, MD (United States); Lawrence Berkeley National Laboratory (LBNL), Berkeley, CA (United States)

The long interspersed element-1 (LINE-1, hereafter L1) retrotransposon has generated nearly one-third of the human genome and serves as an active source of genetic diversity and human disease. L1 spreads through a mechanism termed target-primed reverse transcription, in which the encoded enzyme (ORF2p) nicks the target DNA to prime reverse transcription of its own or non-self RNAs. Here we purified full-length L1 ORF2p and biochemically reconstituted robust target-primed reverse transcription with template RNA and target-site DNA. We report cryo-electron microscopy structures of the complete human L1 ORF2p bound to structured template RNAs and initiating cDNA synthesis. The template polyadenosine tract is recognized in a sequence-specific manner by five distinct domains. Among them, an RNA-binding domain bends the template backbone to allow engagement of an RNA hairpin stem with the L1 ORF2p C-terminal segment. Moreover, structure and biochemical reconstitutions demonstrate an unexpected target-site requirement: L1 ORF2p relies on upstream single-stranded DNA to position the adjacent duplex in the endonuclease active site for nicking of the longer DNA strand, with a single nick generating a staggered DNA break. Our research provides insights into the mechanism of ongoing transposition in the human genome and informs the engineering of retrotransposon proteins for gene therapy.

View Accepted Manuscript (DOE)

Cite

Export

Save

Research Organization:: Lawrence Berkeley National Laboratory (LBNL), Berkeley, CA (United States)

Sponsoring Organization:: USDOE; National Institutes of Health (NIH)

Grant/Contract Number:: AC02-05CH11231

OSTI ID:: 2470973

Journal Information:: Nature (London), Journal Name: Nature (London) Journal Issue: 7997 Vol. 626; ISSN 0028-0836

Publisher:: Nature Publishing GroupCopyright Statement

Country of Publication:: United States

Language:: English

References (61)

UCSF ChimeraX : Structure visualization for researchers, educators, and developers Pettersen, Eric F.; Goddard, Thomas D.; Huang, Conrad C. Protein Science, Vol. 30, Issue 1 https://doi.org/10.1002/pro.3943	journal	October 2020
DALI shines a light on remote homologs: One hundred discoveries Holm, Liisa; Laiho, Aleksi; Törönen, Petri Protein Science, Vol. 32, Issue 1 https://doi.org/10.1002/pro.4519	journal	December 2022
Human L1 Retrotransposon Encodes a Conserved Endonuclease Required for Retrotransposition Feng, Qinghua; Moran, John V.; Kazazian, Haig H. Cell, Vol. 87, Issue 5 https://doi.org/10.1016/S0092-8674(00)81997-2	journal	November 1996
High Frequency Retrotransposition in Cultured Mammalian Cells Moran, John V.; Holmes, Susan E.; Naas, Thierry P. Cell, Vol. 87, Issue 5 https://doi.org/10.1016/S0092-8674(00)81998-4	journal	November 1996
Affinity Proteomics Reveals Human Host Factors Implicated in Discrete Stages of LINE-1 Retrotransposition Taylor, Martin S.; LaCava, John; Mita, Paolo Cell, Vol. 155, Issue 5 https://doi.org/10.1016/j.cell.2013.10.021	journal	November 2013
Genome-wide de novo L1 Retrotransposition Connects Endonuclease Activity with Replication Flasch, Diane A.; Macia, Ángela; Sánchez, Laura Cell, Vol. 177, Issue 4 https://doi.org/10.1016/j.cell.2019.02.050	journal	May 2019
Cryo-EM Structures of a Group II Intron Reverse Splicing into DNA Haack, Daniel B.; Yan, Xiaodong; Zhang, Cheng Cell, Vol. 178, Issue 3 https://doi.org/10.1016/j.cell.2019.06.035	journal	July 2019
Structural RNA components supervise the sequential DNA cleavage in R2 retrotransposon Deng, Pujuan; Tan, Shun-Qing; Yang, Qi-Yu Cell, Vol. 186, Issue 13 https://doi.org/10.1016/j.cell.2023.05.032	journal	June 2023
The carboxy-terminal segment of the human LINE-1 ORF2 protein is involved in RNA binding Piskareva, Olga; Ernst, Christina; Higgins, Niamh FEBS Open Bio, Vol. 3, Issue 1 https://doi.org/10.1016/j.fob.2013.09.005	journal	January 2013
CTFFIND4: Fast and accurate defocus estimation from electron micrographs Rohou, Alexis; Grigorieff, Nikolaus Journal of Structural Biology, Vol. 192, Issue 2 https://doi.org/10.1016/j.jsb.2015.08.008	journal	November 2015
Retrotransposition and Crystal Structure of an Alu RNP in the Ribosome-Stalling Conformation Ahl, Valentina; Keller, Heiko; Schmidt, Steffen Molecular Cell, Vol. 60, Issue 5 https://doi.org/10.1016/j.molcel.2015.10.003	journal	December 2015
A 3′ Poly(A) Tract Is Required for LINE-1 Retrotransposition Doucet, Aurélien J.; Wilusz, Jeremy E.; Miyoshi, Tomoichiro Molecular Cell, Vol. 60, Issue 5 https://doi.org/10.1016/j.molcel.2015.10.012	journal	December 2015
Structure of a Thermostable Group II Intron Reverse Transcriptase with Template-Primer and Its Functional and Evolutionary Implications Stamos, Jennifer L.; Lentzsch, Alfred M.; Lambowitz, Alan M. Molecular Cell, Vol. 68, Issue 5 https://doi.org/10.1016/j.molcel.2017.10.024	journal	December 2017
The Landscape of L1 Retrotransposons in the Human Genome Is Shaped by Pre-insertion Sequence Biases and Post-insertion Selection Sultana, Tania; van Essen, Dominic; Siol, Oliver Molecular Cell, Vol. 74, Issue 3 https://doi.org/10.1016/j.molcel.2019.02.036	journal	May 2019
Crystal Structure of the Targeting Endonuclease of the Human LINE-1 Retrotransposon Weichenrieder, Oliver; Repanas, Kostas; Perrakis, Anastassis Structure, Vol. 12, Issue 6 https://doi.org/10.1016/j.str.2004.04.011	journal	June 2004
Role of poly(A) tail length in Alu retrotransposition Dewannieux, M.; Heidmann, T. Genomics, Vol. 86, Issue 3 https://doi.org/10.1016/j.ygeno.2005.05.009	journal	September 2005
Targeting of Human Retrotransposon Integration Is Directed by the Specificity of the L1 Endonuclease for Regions of Unusual DNA Structure Cost, Gregory J.; Boeke, Jef D. Biochemistry, Vol. 37, Issue 51 https://doi.org/10.1021/bi981858s	journal	December 1998
Initial sequencing and analysis of the human genome Lander, Eric S.; Linton, Lauren M. Nature, Vol. 409, Issue 6822, p. 860-921 https://doi.org/10.1038/35057062	journal	February 2001
Human LINE retrotransposons generate processed pseudogenes Esnault, Cécile; Maestre, Joël; Heidmann, Thierry Nature Genetics, Vol. 24, Issue 4 https://doi.org/10.1038/74184	journal	April 2000
Somatic retrotransposition alters the genetic landscape of the human brain Baillie, J. Kenneth; Barnett, Mark W.; Upton, Kyle R. Nature, Vol. 479, Issue 7374 https://doi.org/10.1038/nature10531	journal	October 2011
LINEs and Alus — the polyA connection Boeke, Jef D. Nature Genetics, Vol. 16, Issue 1 https://doi.org/10.1038/ng0597-6	journal	May 1997
LINE-mediated retrotransposition of marked Alu sequences Dewannieux, Marie; Esnault, Cécile; Heidmann, Thierry Nature Genetics, Vol. 35, Issue 1 https://doi.org/10.1038/ng1223	journal	August 2003
cryoSPARC: algorithms for rapid unsupervised cryo-EM structure determination Punjani, Ali; Rubinstein, John L.; Fleet, David J. Nature Methods, Vol. 14, Issue 3 https://doi.org/10.1038/nmeth.4169	journal	February 2017
MotionCor2: anisotropic correction of beam-induced motion for improved cryo-electron microscopy Zheng, Shawn Q.; Palovcak, Eugene; Armache, Jean-Paul Nature Methods, Vol. 14, Issue 4 https://doi.org/10.1038/nmeth.4193	journal	February 2017
Crystal structures of a group II intron maturase reveal a missing link in spliceosome evolution Zhao, Chen; Pyle, Anna Marie Nature Structural & Molecular Biology, Vol. 23, Issue 6 https://doi.org/10.1038/nsmb.3224	journal	May 2016
G-quadruplex structures within the 3′ UTR of LINE-1 elements stimulate retrotransposition Sahakyan, Aleksandr B.; Murat, Pierre; Mayer, Clemens Nature Structural & Molecular Biology, Vol. 24, Issue 3 https://doi.org/10.1038/nsmb.3367	journal	January 2017
Cis-preferential LINE-1 reverse transcriptase activity in ribonucleoprotein particles Kulpa, Deanna A.; Moran, John V. Nature Structural & Molecular Biology, Vol. 13, Issue 7 https://doi.org/10.1038/nsmb1107	journal	June 2006
Prime editing for precise and highly versatile genome manipulation Chen, Peter J.; Liu, David R. Nature Reviews Genetics, Vol. 24, Issue 3 https://doi.org/10.1038/s41576-022-00541-1	journal	November 2022
Structure of human telomerase holoenzyme with bound telomeric DNA Ghanim, George E.; Fountain, Adam J.; van Roon, Anne-Marie M. Nature, Vol. 593, Issue 7859 https://doi.org/10.1038/s41586-021-03415-4	journal	April 2021
Highly accurate protein structure prediction with AlphaFold Jumper, John; Evans, Richard; Pritzel, Alexander Nature https://doi.org/10.1038/s41586-021-03819-2	journal	July 2021
Precise genome editing across kingdoms of life using retron-derived DNA Lopez, Santiago C.; Crawford, Kate D.; Lear, Sierra K. Nature Chemical Biology, Vol. 18, Issue 2 https://doi.org/10.1038/s41589-021-00927-y	journal	December 2021
Targeted insertion of large genetic payloads using cas directed LINE-1 reverse transcriptase Manoj, Femila; Tai, Laura W.; Wang, Katelyn Sun Mi Scientific Reports, Vol. 11, Issue 1 https://doi.org/10.1038/s41598-021-03130-0	journal	December 2021
SPHIRE-crYOLO is a fast and accurate fully automated particle picker for cryo-EM Wagner, Thorsten; Merino, Felipe; Stabrin, Markus Communications Biology, Vol. 2, Issue 1 https://doi.org/10.1038/s42003-019-0437-z	journal	June 2019
Mobile DNA in Health and Disease Kazazian, Haig H.; Moran, John V. New England Journal of Medicine, Vol. 377, Issue 4 https://doi.org/10.1056/NEJMra1510092	journal	July 2017
Hot L1s account for the bulk of retrotransposition in the human population Brouha, B.; Schustak, J.; Badge, R. M. Proceedings of the National Academy of Sciences, Vol. 100, Issue 9 https://doi.org/10.1073/pnas.0831042100	journal	April 2003
Sequence patterns indicate an enzymatic involvement in integration of mammalian retroposons Jurka, J. Proceedings of the National Academy of Sciences, Vol. 94, Issue 5 https://doi.org/10.1073/pnas.94.5.1872	journal	March 1997
Bacterial Retrons Enable Precise Gene Editing in Human Cells Zhao, Bin; Chen, Shi-An A.; Lee, Jiwoo The CRISPR Journal, Vol. 5, Issue 1 https://doi.org/10.1089/crispr.2021.0065	journal	February 2022
Human L1 element target-primed reverse transcription in vitro Cost, G. J. The EMBO Journal, Vol. 21, Issue 21 https://doi.org/10.1093/emboj/cdf592	journal	November 2002
The human LINE-1 reverse transcriptase:effect of deletions outside the common reverse transcriptase domain Clements, A. Nucleic Acids Research, Vol. 26, Issue 15 https://doi.org/10.1093/nar/26.15.3528	journal	August 1998
Structural dissection of sequence recognition and catalytic mechanism of human LINE-1 endonuclease Miller, Ian; Totrov, Max; Korotchkina, Lioubov Nucleic Acids Research, Vol. 49, Issue 19 https://doi.org/10.1093/nar/gkab826	journal	September 2021
UniProt: the Universal Protein Knowledgebase in 2023 Nucleic Acids Research, Vol. 51, Issue D1 https://doi.org/10.1093/nar/gkac1052	journal	November 2022
Mapping the LINE1 ORF1 protein interactome reveals associated inhibitors of human retrotransposition Goodier, John L.; Cheung, Ling E.; Kazazian, Haig H. Nucleic Acids Research, Vol. 41, Issue 15 https://doi.org/10.1093/nar/gkt512	journal	June 2013
Active Alu retrotransposons in the human genome Bennett, E. A.; Keller, H.; Mills, R. E. Genome Research, Vol. 18, Issue 12 https://doi.org/10.1101/gr.081737.108	journal	October 2008
PHENIX: a comprehensive Python-based system for macromolecular structure solution Adams, Paul D.; Afonine, Pavel V.; Bunkóczi, Gábor Acta Crystallographica Section D Biological Crystallography, Vol. 66, Issue 2, p. 213-221 https://doi.org/10.1107/S0907444909052925	journal	January 2010
Features and development of Coot Emsley, P.; Lohkamp, B.; Scott, W. G. Acta Crystallographica Section D Biological Crystallography, Vol. 66, Issue 4 https://doi.org/10.1107/S0907444910007493	journal	March 2010
Reverse transcriptase encoded by a human transposable element Mathias, S.; Scott, A.; Kazazian, H. Science, Vol. 254, Issue 5039 https://doi.org/10.1126/science.1722352	journal	December 1991
Repetitive DNA in disease Burns, Kathleen H. Science, Vol. 376, Issue 6591 https://doi.org/10.1126/science.abl7399	journal	April 2022
Structures of a mobile intron retroelement poised to attack its structured DNA target Chung, Kevin; Xu, Ling; Chai, Pengxin Science, Vol. 378, Issue 6620 https://doi.org/10.1126/science.abq2844	journal	November 2022
Structure of the R2 non-LTR retrotransposon initiating target-primed reverse transcription Wilkinson, Max E.; Frangieh, Chris J.; Macrae, Rhiannon K. Science, Vol. 380, Issue 6642 https://doi.org/10.1126/science.adg7883	journal	April 2023
Nucleic Acid Chaperone Activity of the ORF1 Protein from the Mouse LINE-1 Retrotransposon Martin, Sandra L.; Bushman, Frederic D. Molecular and Cellular Biology, Vol. 21, Issue 2 https://doi.org/10.1128/MCB.21.2.467-475.2001	journal	January 2001
Human L1 Retrotransposition: cisPreference versus trans Complementation Wei, Wei; Gilbert, Nicolas; Ooi, Siew Loon Molecular and Cellular Biology, Vol. 21, Issue 4 https://doi.org/10.1128/MCB.21.4.1429-1439.2001	journal	February 2001
Non-long terminal repeat (non-LTR) retrotransposons: mechanisms, recent developments, and unanswered questions Han, Jeffrey S. Mobile DNA, Vol. 1, Issue 1 https://doi.org/10.1186/1759-8753-1-15	journal	January 2010
Alu elements: know the SINEs Deininger, Prescott Genome Biology, Vol. 12, Issue 12 https://doi.org/10.1186/gb-2011-12-12-236	journal	January 2011
Roles for retrotransposon insertions in human disease Hancks, Dustin C.; Kazazian, Haig H. Mobile DNA, Vol. 7, Issue 1 https://doi.org/10.1186/s13100-016-0065-9	journal	May 2016
The RNA Polymerase Dictates ORF1 Requirement and Timing of LINE and SINE Retrotransposition Kroutter, Emily N.; Belancio, Victoria P.; Wagstaff, Bradley J. PLoS Genetics, Vol. 5, Issue 4 https://doi.org/10.1371/journal.pgen.1000458	journal	April 2009
The Zinc-Finger Antiviral Protein ZAP Inhibits LINE and Alu Retrotransposition Moldovan, John B.; Moran, John V. PLOS Genetics, Vol. 11, Issue 5 https://doi.org/10.1371/journal.pgen.1005121	journal	May 2015
Comprehensive Scanning Mutagenesis of Human Retrotransposon LINE-1 Identifies Motifs Essential for Function Adney, Emily M.; Ochmann, Matthias T.; Sil, Srinjoy Genetics, Vol. 213, Issue 4 https://doi.org/10.1534/genetics.119.302601	journal	December 2019
New Understanding of the Relevant Role of LINE-1 Retrotransposition in Human Disease and Immune Modulation Zhang, Xiao; Zhang, Rui; Yu, Jinpu Frontiers in Cell and Developmental Biology, Vol. 8 https://doi.org/10.3389/fcell.2020.00657	journal	August 2020
LINE-1 protein localization and functional dynamics during the cell cycle Mita, Paolo; Wudzinska, Aleksandra; Sun, Xiaoji eLife, Vol. 7 https://doi.org/10.7554/eLife.30058	journal	January 2018
Dissection of affinity captured LINE-1 macromolecular complexes Taylor, Martin S.; Altukhov, Ilya; Molloy, Kelly R. eLife, Vol. 7 https://doi.org/10.7554/eLife.30094	journal	January 2018
New tools for automated high-resolution cryo-EM structure determination in RELION-3 Zivanov, Jasenko; Nakane, Takanori; Forsberg, Björn O. eLife, Vol. 7 https://doi.org/10.7554/eLife.42166	journal	November 2018

Similar Records

Structural dissection of sequence recognition and catalytic mechanism of human LINE-1 endonuclease

Journal Article · 2021 · Nucleic Acids Research · OSTI ID:1821946

Miller, Ian; Totrov, Max; Korotchkina, Lioubov; +3 more

Identification and characterization of small molecule inhibitors of the LINE-1 retrotransposon endonuclease

Journal Article · 2024 · Nature Communications · OSTI ID:2469583

D’Ordine, Alexandra M.; Jogl, Gerwald; Sedivy, John M.

Analysis of the 5{prime} junctions of R2 insertions with the 28S gene: Implications for non-LTR retrotransposition

Journal Article · 1996 · Genetics · OSTI ID:539427

George, J A; Burke, W D; Eickbush, T H

Related Subjects

59 BASIC BIOLOGICAL SCIENCES
Cryoelectron microscopy
RNA
Science & Technology - Other Topics
Transposition

Title: Template and target-site recognition by human LINE-1 in retrotransposition

Citation Formats

References (61)

Similar Records

Related Subjects