The long interspersed nuclear element-1 (L1) makes up nearly two-thirds of the human genome and serves as an active source of genetic diversity and human diseases. L1 spreads through a mechanism called target-primed reverse transcription (TPRT), where the encoded enzyme (ORF2p) cleaves the target DNA to stimulate reverse transcription of its own DNA or self/nonself RNA. Here, we have purified full-length ORF2p and reconstituted biologically active TPRT using template RNA and a target site. We present cryo-electron microscopy structures of human ORF2p bound to structured template RNA and initiate cDNA synthesis. The pathway of the template polyadenosine is specifically recognized for sequencing by five distinct domains. Among these, new domains for RNA binding bend the template backbone to allow tethering of the RNA hairpin stem with the human polar portion of ORF2p. Furthermore, the structures and biological reconstitutions reveal unexpected requirements for the target site: ORF2p relies on advanced single-stranded DNA to position the adjacent duplex in the active nuclease site to cleave the longer strand of DNA, generating a single-stranded break in the subsequent DNA. Our work provides key insights into the mechanism of ongoing retrotransposition in the human genome and informs the engineering of reverse transcribing gene proteins.
Abstract
The long interspersed nuclear element-1 (L1) makes up nearly two-thirds of the human genome and serves as an active source of genetic diversity and human diseases. L1 spreads through a mechanism called target-primed reverse transcription (TPRT), where the encoded enzyme (ORF2p) cleaves the target DNA to stimulate reverse transcription of its own DNA or self/nonself RNA. Here, we have purified full-length ORF2p and reconstituted biologically active TPRT using template RNA and a target site. We present cryo-electron microscopy structures of human ORF2p bound to structured template RNA and initiate cDNA synthesis. The pathway of the template polyadenosine is specifically recognized for sequencing by five distinct domains. Among these, new domains for RNA binding bend the template backbone to allow tethering of the RNA hairpin stem with the human polar portion of ORF2p. Furthermore, the structures and biological reconstitutions reveal unexpected requirements for the target site: ORF2p relies on advanced single-stranded DNA to position the adjacent duplex in the active nuclease site to cleave the longer strand of DNA, generating a single-stranded break in the subsequent DNA. Our work provides key insights into the mechanism of ongoing retrotransposition in the human genome and informs the engineering of reverse transcribing gene proteins.
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Akanaksha Thawani, Alfredo José Florez Ariza, Eva Nogales, Kathleen Collins
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Correspondence to Akanaksha Thawani, Eva Nogales, or Kathleen Collins.
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This file contains Supplementary Fig. 1 and Supplementary Table 1. Supplementary Fig. 1: Untrimmed gels of Fig. 1c, 1d, 2c, 2g, 3d, 3f, 4b-e, and Extended Data Figs. 1a, 1c, 1d, 5e, 7a, 7b, 8, and 10b. Supplementary Table 1: All DNA sequences used in this study.
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Thawani, A., Ariza, A.J.F., Nogales, E. et al. Template and target site recognition by human LINE-1 in retrotransposition. Nature (2023). https://doi.org/10.1038/s41586-023-06933-5
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