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

The retrotransposon Long Interspersed Element-1 (L1) contains adenosine rich open reading frames (ORFs), a characteristic that limits its expression in mammalian cells. A synthetic mouse L1 (smL1) with ORF adenosine content decreased from 40% to 26% showed increased mRNA expression and retrotransposed far more efficiently than the native parental element, L1spa (1). 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) which could take on a G-quadruplex secondary structure. We find that the combined effect of the altered ORF1p amino acid sequence and the GRR 3'UTR deletion, rather than synthetic ORF sequences, accounts for the increase in smL1 retrotransposition efficiency over L1spa. We determine that inclusion of the GRR influences natural mouse L1 retrotransposition in a position-dependent manner that does not involve premature polyadenylation of L1 mRNA transcripts or alteration of L1 insertion structures. Instead, we present evidence that the position-dependent effect of the GRR on natural mouse retrotransposition occurs through modulation of L1 ORF1p abundance. In contrast, smL1 retrotransposition is refractory to the inhibitory effect of the GRR, likely owing to highly efficient protein expression from synthetic ORF sequences. Taken together, our results prompt further investigation of the relationship between the GRR sequence and L1 protein expression and demonstrate that the arrangement of L1 nucleotide sequences within reporter constructs can influence L1 expression and engineered retrotransposition efficiency.