L1 retrotransposition: The snap-velcro model and its consequences.

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.

The specificity and flexibility of l1 reverse transcription priming at imperfect T-tracts.

L1 retrotransposons have a prominent role in reshaping mammalian genomes. To replicate, the L1 ribonucleoprotein particle (RNP) first uses its endonuclease (EN) to nick the genomic DNA. The newly generated DNA end is subsequently used as a primer to initiate reverse transcription within the L1 RNA poly(A) tail, a process known as target-primed reverse transcription (TPRT). Prior studies demonstrated that most L1 insertions occur into sequences related to the L1 EN consensus sequence (degenerate 5'-TTTT/A-3' sites) and frequently preceded by imperfect T-tracts. However, it is currently unclear whether--and to which degree--the liberated 3'-hydroxyl extremity on the genomic DNA needs to be accessible and complementary to the poly(A) tail of the L1 RNA for efficient priming of reverse transcription. Here, we employed a direct assay for the initiation of L1 reverse transcription to define the molecular rules that guide this process. First, efficient priming is detected with as few as 4 matching nucleotides at the primer 3' end. Second, L1 RNP can tolerate terminal mismatches if they are compensated within the 10 last bases of the primer by an increased number of matching nucleotides. All terminal mismatches are not equally detrimental to DNA extension, a C being extended at higher levels than an A or a G. Third, efficient priming in the context of duplex DNA requires a 3' overhang. This suggests the possible existence of additional DNA processing steps, which generate a single-stranded 3' end to allow L1 reverse transcription. Based on these data we propose that the specificity of L1 reverse transcription initiation contributes, together with the specificity of the initial EN cleavage, to the distribution of new L1 insertions within the human genome.

RNA-mediated interference and reverse transcription control the persistence of RNA viruses in the insect model Drosophila.

How persistent viral infections are established and maintained is widely debated and remains poorly understood. We found here that the persistence of RNA viruses in Drosophila melanogaster was achieved through the combined action of cellular reverse-transcriptase activity and the RNA-mediated interference (RNAi) pathway. Fragments of diverse RNA viruses were reverse-transcribed early during infection, which resulted in DNA forms embedded in retrotransposon sequences. Those virus-retrotransposon DNA chimeras produced transcripts processed by the RNAi machinery, which in turn inhibited viral replication. Conversely, inhibition of reverse transcription hindered the appearance of chimeric DNA and prevented persistence. Our results identify a cooperative function for retrotransposons and antiviral RNAi in the control of lethal acute infection for the establishment of viral persistence.