Figure 4. Reverse transcription by L1 ORF2p increases A-tail length of new Alu inserts and helps maintain viable Alu source elements over evolutionary time.

A. A-tail expansion by the L1 ORF2p endonuclease. We propose a model where expansion of the A-tail occurs early during reverse transcription by the L1 ORF2p due to an unstable interaction between the Alu RNA (green) and the cDNA. A-tail expansion may occur through either “looping out” of the cDNA or rounds of dissociations and re-annealing between the two molecules causing priming and reverse transcription to reinitiate, leading to an increase in size. Note that the “looping out” of the RNA would cause a contraction of the A-tail instead of a lengthening. Although speculative, we propose that the RNA folding may be constricted due to interaction with proteins such as polyA binding protein (PABP, shown as gray circles). The interaction with the potentially bound proteins may also prevent priming from occurring at the most internal adenosines of the A-tail. The nascent cDNA strand (purple) initially only provides a weak interaction allowing for slippage or dissociation to occur. As the cDNA lengthens (orange), the additional hydrogen bonding between molecules eventually stabilizes the process. Depending on where the re-initiation of reverse transcription occurs during slippage, the non-adenosine nucleotides can be duplicated in the cDNA sequence as shown in our model. The bottom panel shows sequences of the A-tail of five recovered Alu clones with duplications of the non-A disruptions (highlighted in gray) that support the model. B. L1 ORF2p maintains active source Alu elements across evolutionary time. We present a model where A-tail expansion of new Alu elements plays an important role in replenishing Alu source genes through time. The two panels depict scenarios with (top) and without (bottom) A-tail expansion. Both scenarios begin with an early active source element (Alu1 activity in red). Through time Alu1 (red) gives rise to a new source element Alu2 (blue), which in turn gives rise to another source element Alu3 (yellow) with differing retrotransposition efficiency. Over time, Alu source elements accumulate inactivating mutations; thus the proliferation of Alu in a given population depends on the generation of new source elements. In the scenario with no A-tail expansion (top panel), new Alu source elements will have shorter A-tails (Alu3-A) and lose the ability to support retrotransposition. Without the possibility of expanding A-tails, extinction may occur. In the alternate scenario (bottom panel), new inserts are introduced with an expanded A-tail. The new Alu will become a source element with an expanded A-tail and generate the next subfamily of Alu inserts. The expansion of the Alu A-tail by the L1 ORF2 plays an important role in the continued genesis of new active source Alu elements within a population.