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

Akanksha Thawani (), Alfredo Jose Florez Ariza, Eva Nogales () and Kathleen Collins ()
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Akanksha Thawani: California Institute for Quantitative Biosciences (QB3)
Alfredo Jose Florez Ariza: California Institute for Quantitative Biosciences (QB3)
Eva Nogales: California Institute for Quantitative Biosciences (QB3)
Kathleen Collins: California Institute for Quantitative Biosciences (QB3)

Nature, 2024, vol. 626, issue 7997, 186-193

Abstract: Abstract 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 disease1. 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 RNAs2. 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.

Date: 2024
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DOI: 10.1038/s41586-023-06933-5

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