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 cancer2,3, autoimmunity4,5 and ageing6,7, 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 production6–8. 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.

CRISPR-Enabled Autonomous Transposable Element (CREATE) for RNA-based gene editing and delivery

“…Similarly, the removal of the entire EN domain (CREATE mRNA ΔEN ) resulted in a marked decrease in editing efficiency, indicating that while the EN domain must be rendered inactive to avert cleavage of the genome at redundant consensus sites recognized by L1 naturally, its complete removal was detrimental to the editing process. This is consistent with the recent reports showing that the EN domain and RT domain of L1 ORF2p are highly integrated structurally and functionally 23,27 .…”

“…1a, step 1). Natural L1 retrotransposition relies on nicking of the non-targeting DNA strand by the EN domain of ORF2p 23,27 . To harness this mechanism, a Cas9 H840A non-target strand nickase is employed to introduce a single strand nick guided by sgRNA1 (Fig 1a , step 2).…”

“…A distinctive feature of L1 is its 'cis preference,' where ORF1p and ORF2p associate with their own mRNA transcript to form a ribonuclear protein complex (RNP) 19 , facilitating the reverse transcription and insertion of the L1 mRNA 20,21 . The endonuclease domain of ORF2p cleaves at a redundant 5'TTTT/AA3' consensus sequence, initiating a target-primed reverse transcription (TPRT) process mediated by the RT domain of ORF2p that converts the L1 mRNA into cDNA and integrates into the genome 22,23 . The ORF2p RT domain was shown to be highly processive, capable of reverse transcription of long RNA sequences 23,24 .…”

“…The endonuclease domain of ORF2p cleaves at a redundant 5'TTTT/AA3' consensus sequence, initiating a target-primed reverse transcription (TPRT) process mediated by the RT domain of ORF2p that converts the L1 mRNA into cDNA and integrates into the genome 22,23 . The ORF2p RT domain was shown to be highly processive, capable of reverse transcription of long RNA sequences 23,24 . However, the consensus motif recognized by the EN domain of L1 ORF2p is found at numerous sites in the genome, precluding its application for targeted, programmable gene editing.…”

p53 mediated regulation of LINE1 retrotransposition derived R-loops

“…L1 encoded ORF2 protein contains two functional domains essential for target-primed reverse transcription: the Endonuclease (EN) domain creates a single-stranded DNA nick for integration, and the Reverse Transcriptase (RT) domain primes complementary DNA synthesis based on the L1mRNA template. The recent structural findings on ORF2p identified domains that shows extensive interactions with RNA templates 24 . The reverse transcription function of ORF2p generates a L1mRNA-cDNA hybrid structure, hence referred to as 'trans R-loop' intermediate structure comprising a free and un-nicked DNA segment, a nicked DNA, 4 the newly synthesized cDNA, and the L1mRNA serving as the template for reverse transcription, probably the four stranded non-canonical form of R-loops.…”