Abstract The retrotransposon Long Interspersed Element 1 (L1) contains adenosine rich ORFs, a characteristic that limits its expression in mammalian cells. A previously developed synthetic mouse L1 (smL1) with ORF adenosine content decreased from 40% to 26% showed higher mRNA expression levels and retrotransposed far more efficiently than the native parental element, L1spa. Here, we observe two nonsynonymous substitutions between the L1spa and smL1 ORF1 sequences, and note that the smL1 3’UTR lacks a conserved guanosine-rich region (GRR). We find that the combined effect of these amino acid changes and the 3’UTR deletion, rather than synthetic ORF sequences, accounts for the dramatic and reproducible increase in smL1 retrotransposition efficiency over L1spa. Furthermore, we demonstrate that the position of the GRR within the L1 reporter construct impacts retrotransposition efficiency. Our results prompt a reevaluation of synthetic L1 activity and suggest that native mouse L1 mobility is frequently underestimated in engineered retrotransposition assays.

Revisiting the impact of synthetic ORF sequences on engineered LINE-1 retrotransposition