Mobile elements comprise close to one half of the mass of the human genome. Only LINE-1 (L1), an autonomous non-Long Terminal Repeat (LTR) retrotransposon, and its non-autonomous partners—such as the retropseudogenes, SVA, and the SINE, Alu—are currently active human retroelements. Experimental evidence shows that Alu retrotransposition depends on L1 ORF2 protein, which has led to the presumption that LINEs and SINEs share the same basic insertional mechanism. Our data demonstrate clear differences in the time required to generate insertions between marked Alu and L1 elements. In our tissue culture system, the process of L1 insertion requires close to 48 hours. In contrast to the RNA pol II-driven L1, we find that pol III transcribed elements (Alu, the rodent SINE B2, and the 7SL, U6 and hY sequences) can generate inserts within 24 hours or less. Our analyses demonstrate that the observed retrotransposition timing does not dictate insertion rate and is independent of the type of reporter cassette utilized. The additional time requirement by L1 cannot be directly attributed to differences in transcription, transcript length, splicing processes, ORF2 protein production, or the ability of functional ORF2p to reach the nucleus. However, the insertion rate of a marked Alu transcript drastically drops when driven by an RNA pol II promoter (CMV) and the retrotransposition timing parallels that of L1. Furthermore, the “pol II Alu transcript” behaves like the processed pseudogenes in our retrotransposition assay, requiring supplementation with L1 ORF1p in addition to ORF2p. We postulate that the observed differences in retrotransposition kinetics of these elements are dictated by the type of RNA polymerase generating the transcript. We present a model that highlights the critical differences of LINE and SINE transcripts that likely define their retrotransposition timing.

Non-long terminal repeat retroelements continue to impact the human genome through cis-activity of long interspersed element-1 (LINE-1 or L1) and trans-mobilization of Alu. Current activity is dominated by modern subfamilies of these elements, leaving behind an evolutionary graveyard of extinct Alu and L1 subfamilies. Because Alu is a nonautonomous element that relies on L1 to retrotranspose, there is the possibility that competition between these elements has driven selection and antagonistic coevolution between Alu and L1. Through analysis of synonymous versus nonsynonymous codon evolution across L1 subfamilies, we find that the C-terminal ORF2 cys domain experienced a dramatic increase in amino acid substitution rate in the transition from L1PA5 to L1PA4 subfamilies. This observation coincides with the previously reported rapid evolution of ORF1 during the same transition period. Ancestral Alu sequences have been previously reconstructed, as their short size and ubiquity have made it relatively easy to retrieve consensus sequences from the human genome. In contrast, creating constructs of extinct L1 copies is a more laborious task. Here, we report our efforts to recreate and evaluate the retrotransposition capabilities of two ancestral L1 elements, L1PA4 and L1PA8 that were active ∼18 and ∼40 Ma, respectively. Relative to the modern L1PA1 subfamily, we find that both elements are similarly active in a cell culture retrotransposition assay in HeLa, and both are able to efficiently trans-mobilize Alu elements from several subfamilies. Although we observe some variation in Alu subfamily retrotransposition efficiency, any coevolution that may have occurred between LINEs and SINEs is not evident from these data. Population dynamics and stochastic variation in the number of active source elements likely play an important role in individual LINE or SINE subfamily amplification. If coevolution also contributes to changing retrotransposition rates and the progression of subfamilies, cell factors are likely to play an important mediating role in changing LINE-SINE interactions over evolutionary time.