Structure of the R2 non-LTR retrotransposon initiating target-primed reverse transcription

Abstract

Non-LTR retrotransposons, or Long Interspersed Nuclear Elements (LINEs), are an abundant class of eukaryotic transposons that insert into genomes by target-primed reverse transcription (TPRT). During TPRT, a target DNA sequence is nicked and primes reverse transcription of the retrotransposon RNA. Here, we report the cryo-electron microscopy structure of the Bombyx mori R2 non-LTR retrotransposon initiating TPRT at its ribosomal DNA target. The target DNA sequence is unwound at the insertion site and recognized by an upstream motif. An extension of the reverse transcriptase (RT) domain recognizes the retrotransposon RNA and guides the 3′ end into the RT active site to template reverse transcription. We used Cas9 to retarget R2 in vitro to non-native sequences, suggesting future use as a reprogrammable RNA-based gene-insertion tool.

Nanopore molecular trajectories of a eukaryotic reverse transcriptase reveal a long-range RNA structure sensing mechanism

“…Our model suggests that bmRT interacts with 11-12 nt of the downstream template (including the stepped-over nucleotide itself, Figure 3B ). Cryo-EM structures of the full length bombyx mori non-LTR R2 retroelement RT shows the formation of a stable complex between the enzyme and a specific hairpin-ssRNA-hairpin-ssRNA structure at the 3’ end of its native mRNA template 24,25 . While this interaction is described to be very specific to the RNA’s 3’ end hairpin structure, it is possible that an extensive bmRT-RNA binding interface, which is not specific to the native RNA hairpin structure, exists.…”

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

“…In addition, insertion at rDNA site may result in reduced expression over time due to rDNA array instability 36 . An attempt to integrate R2 elements with Cas9 for RNA-guided reverse transcription and integration was unsuccessful to achieve complete cDNA synthesis and integration 25 . Exploiting the natural replication mechanisms of an engineered L1 element, CREATE achieved an integration efficiency of approximately 1.5% in multiple mammalian cell lines, inserting a 1.1 kb GFP cassette without detectable off-target integration in multiple genomic sites.…”

“…However, in its native form, the L1 EN domain lacks sequence specificity, precluding its application in targeted, programmable gene editing. A recent study attempting to combine Cas9 with R2 retroelement via a direct fusion approach was unable to achieve complete retrotransposition and integration ( 23 ).…”

“…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. A recent study attempting to combine Cas9 with another retroelement, the R2 retroelement via a direct fusion approach was unable to achieve complete retrotransposition and integration 25 .…”

State‐of‐the‐art in transposable element modulation affected by drugs in malignant prostatic cancer cells

“…Then, the RT identifies the retrotransposon RNA and leads its 3′-end inside the catalytic site to finalize the transposition. 7,[25][26][27][28] Concerning the tools used for characterizing TEs, the development and implementation of Repbase, a database containing eukaryotic repeat sequences, have facilitated the identification of different TE types (Helitron, Polinton, Ginger, and SINEU) and improved their classification based on bioinformatics analysis of sequence, protein domain composition, and structural hallmarks, also enhancing their genomic detection. 11 Twenty recently identified TE subfamilies with regulatory potential have been discovered using a novel statistical method based on overlapping sequences, which enables the exclusion of false positives found in public databases.…”