L1 retrotransposition: The snap-velcro model and its consequences

Abstract

LINE-1 (L1) elements are the only active and autonomous transposable elements in humans. The core retrotransposition machinery is a ribonucleoprotein particle (RNP) containing the L1 mRNA, with endonuclease and reverse transcriptase activities. It initiates reverse transcription directly at genomic target sites upon endonuclease cleavage. Recently, using a direct L1 extension assay (DLEA), we systematically tested the ability of native L1 RNPs to extend DNA substrates of various sequences and structures. We deduced from these experiments the general rules guiding the initiation of L1 reverse transcription, referred to as the snap-velcro model. In this model, L1 target choice is not only mediated by the sequence specificity of the endonuclease, but also through base-pairing between the L1 mRNA and the target site, which permits the subsequent L1 reverse transcription step. In addition, L1 reverse transcriptase efficiently primes L1 DNA synthesis only when the 3' end of the DNA substrate is single-stranded, suggesting so-far unrecognized DNA processing steps at the integration site.

Keywords: L1; LINE-1; TPRT; endonuclease; non-LTR retrotransposon; poly(A) tail; reverse transcriptase; reverse transcription; target-primed reverse transcription.

Figure 1. The L1 life-cycle. L1 replication starts by the transcription of a bicistronic mRNA (A). The L1 RNA is exported to the cytoplasm (B). ORF1p and ORF2p proteins are translated and bind to the L1 RNA to form L1 ribonucleoprotein particles (RNP) (C). The L1 RNP is imported into the nucleus (D). Integration and reverse transcription occur at the genomic target site. First, the L1 endonuclease (EN) activity nicks the target DNA (red arrowhead, E). Then, the L1 reverse transcriptase (RT) initiates the reverse transcription of L1 RNA (black arrowhead, F). The mechanisms involved in the final steps of this process and the resolution of the integration are unresolved yet (G). Partial reverse transcription can lead to 5′-truncated L1 copies.

Figure 2. Features of the snap-velcro model of L1 reverse transcription priming. (A) Reverse transcription priming only occurs if the DNA substrate is single-stranded. (B) Reverse transcription priming requires base-pairing between the L1 RNA (pink) poly(A) tail and the target-site DNA (green). The snap (bold green) corresponds to the last 4 nucleotides at the 3′ of the DNA primer. The velcro (light green) contains the 6 bases upstream of the snap. The snap is considered as closed if 4 nucleotides are T. The velcro is tightly fastened if the position-weighted T-density is superior to 0.5 (see ref. for the detailed numerical model). The snap-velcro status predicts the efficiency of L1 reverse transcription priming (green arrow).

Figure 3. Hypothetical mechanisms allowing L1 RNA base-pairing with target-site DNA. (A) ORF2p dimerization leads to simultaneous staggered cuts through its EN activity. The resulting extremities have 3′ overhangs, which can anneal to the L1 RNA and prime L1 cDNA synthesis. (B) L1 EN initially starts with a single cut, but a DNA-dependent helicase unwinds the target site DNA strands, enabling L1 cDNA synthesis. (C) Upon double-strand DNA break, DNA repair factors resect these ends and generate 3′ overhangs. These new extremities not necessarily end with Ts as for EN sites. Consequently, base-pairing generally occurs at internal sites within the L1 RNA which show spurious matches with the damaged site. (D) Telomeres naturally end with 3′ overhangs. Red arrowhead, EN cut; green, cDNA; pink, L1 RNA.