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Structures, functions and adaptations of the human LINE-1 ORF2 protein.

ABSTRACT: The LINE-1 (L1) retrotransposon is an ancient genetic parasite that has written around one-third of the human genome through a 'copy and paste' mechanism catalysed by its multifunctional enzyme, open reading frame 2 protein (ORF2p)¹. ORF2p reverse transcriptase (RT) and endonuclease activities have been implicated in the pathophysiology of cancer^2,3, autoimmunity^4,5 and ageing^6,7, making ORF2p a potential therapeutic target. However, a lack of structural and mechanistic knowledge has hampered efforts to rationally exploit it. We report structures of the human ORF2p 'core' (residues 238-1061, including the RT domain) by X-ray crystallography and cryo-electron microscopy in several conformational states. Our analyses identified two previously undescribed folded domains, extensive contacts to RNA templates and associated adaptations that contribute to unique aspects of the L1 replication cycle. Computed integrative structural models of full-length ORF2p show a dynamic closed-ring conformation that appears to open during retrotransposition. We characterize ORF2p RT inhibition and reveal its underlying structural basis. Imaging and biochemistry show that non-canonical cytosolic ORF2p RT activity can produce RNA:DNA hybrids, activating innate immune signalling through cGAS/STING and resulting in interferon production^6-8. In contrast to retroviral RTs, L1 RT is efficiently primed by short RNAs and hairpins, which probably explains cytosolic priming. Other biochemical activities including processivity, DNA-directed polymerization, non-templated base addition and template switching together allow us to propose a revised L1 insertion model. Finally, our evolutionary analysis demonstrates structural conservation between ORF2p and other RNA- and DNA-dependent polymerases. We therefore provide key mechanistic insights into L1 polymerization and insertion, shed light on the evolutionary history of L1 and enable rational drug development targeting L1.

SUBMITTER: Baldwin ET

PROVIDER: S-EPMC10830420 | biostudies-literature | 2024 Feb

REPOSITORIES: biostudies-literature

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Structures, functions and adaptations of the human LINE-1 ORF2 protein.

Baldwin Eric T ET van Eeuwen Trevor T Hoyos David D Zalevsky Arthur A Tchesnokov Egor P EP Sánchez Roberto R Miller Bryant D BD Di Stefano Luciano H LH Ruiz Francesc Xavier FX Hancock Matthew M Işik Esin E Mendez-Dorantes Carlos C Walpole Thomas T Nichols Charles C Wan Paul P Riento Kirsi K Halls-Kass Rowan R Augustin Martin M Lammens Alfred A Jestel Anja A Upla Paula P Xibinaku Kera K Congreve Samantha S Hennink Maximiliaan M Rogala Kacper B KB Schneider Anna M AM Fairman Jennifer E JE Christensen Shawn M SM Desrosiers Brian B Bisacchi Gregory S GS Saunders Oliver L OL Hafeez Nafeeza N Miao Wenyan W Kapeller Rosana R Zaller Dennis M DM Sali Andrej A Weichenrieder Oliver O Burns Kathleen H KH Götte Matthias M Rout Michael P MP Arnold Eddy E Greenbaum Benjamin D BD Romero Donna L DL LaCava John J Taylor Martin S MS

Nature 20231214 7997

The LINE-1 (L1) retrotransposon is an ancient genetic parasite that has written around one-third of the human genome through a 'copy and paste' mechanism catalysed by its multifunctional enzyme, open reading frame 2 protein (ORF2p)1. ORF2p reverse transcriptase (RT) and endonuclease activities have been implicated in the pathophysiology of cancer2,3, autoimmunity4,5 and ageing6,7, making ORF2p a potential therapeutic target. However, a lack of structur ...[more]

PMID: 38096902

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Project description:BACKGROUND:Approximately 17 % of the human genome is comprised of the Long INterspersed Element-1 (LINE-1 or L1) retrotransposon, the only currently active autonomous family of retroelements. Though L1 elements have helped to shape mammalian genome evolution over millions of years, L1 activity can also be mutagenic and result in human disease. L1 expression has the potential to contribute to genomic instability via retrotransposition and DNA double-strand breaks (DSBs). Additionally, L1 is responsible for structural genomic variations induced by other transposable elements such as Alu and SVA, which rely on the L1 ORF2 protein for their propagation. Most of the genomic damage associated with L1 activity originates with the endonuclease domain of the ORF2 protein, which nicks the DNA in preparation for target-primed reverse transcription. RESULTS:Bioinformatic analysis of full-length L1 loci residing in the human genome identified numerous mutations in the amino acid sequence of the ORF2 endonuclease domain. Some of these mutations were found in residues which were predicted to be phosphorylation sites for cellular kinases. We mutated several of these putative phosphorylation sites in the ORF2 endonuclease domain and investigated the effect of these mutations on the function of the full-length ORF2 protein and the endonuclease domain (ENp) alone. Most of the single and multiple point mutations that were tested did not significantly impact expression of the full-length ORF2p, or alter its ability to drive Alu retrotransposition. Similarly, most of those same mutations did not significantly alter expression of ENp, or impair its ability to induce DNA damage and cause toxicity. CONCLUSIONS:Overall, our data demonstrate that the full-length ORF2p or the ENp alone can tolerate several specific single and multiple point mutations in the endonuclease domain without significant impairment of their ability to support Alu mobilization or induce DNA damage, respectively.

Dataset Information

Structures, functions and adaptations of the human LINE-1 ORF2 protein.

Publications

Structures, functions and adaptations of the human LINE-1 ORF2 protein.

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