Functional analyses identify the L1 ORF2p Cryptic domain as a hub for intramolecular interfaces required for retrotransposition.

Endonuclease (EN) and reverse transcriptase (RT) functions of Long Interspersed Element-1 (L1) ORF2p are required for DNA-nicking and complementary DNA (cDNA) synthesis during retrotransposition. These domains are homologous to cellular/viral proteins and are well-studied. The ORF2p Cryptic region has no known homologues, is evolutionarily conserved among L1-like elements, and is required for retrotransposition through an unknown mechanism. While ORF2p structures establish Cryptic interactions with the EN, RT, and C-terminal domains (CTD), functional importance of these interactions remains unclear. We examine functional contributions of these intramolecular interactions in retrotransposition through mutagenesis, co-immunoprecipitation, cDNA synthesis, RNA binding, retrotransposition, cytotoxicity, and molecular modeling analyses. We demonstrate that Cryptic functions as an independent domain capable of reestablishing functional intramolecular interactions in trans to rescue cDNA synthesis. The Cryptic:RT interface is required for cDNA initiation/elongation and may also affect EN function in retrotransposition, while eliminating ORF2p toxicity. In contrast, the Cryptic:CTD interface is not required for first strand cDNA synthesis but affects retrotransposition and increases ORF2p toxicity. Overall, intramolecular interactions involving Cryptic are required for multiple steps of retrotransposition and propose Cryptic as a nonenzymatic structural domain and a promising target for therapeutic interventions in cancers with upregulated L1 expression.