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. Furthermore, we demonstrate that the presence and position of the GRR within the L1 reporter construct impact mouse L1 ORF1p expression and retrotransposition efficiency. Our results prompt a reevaluation of synthetic L1 activity and suggest that the manner in which L1 sequences are cloned into engineered reporter vectors has, in some cases, resulted in an underestimation of native mouse L1 retrotransposition efficiency. Author Summary L1 retrotransposons are mobile DNA elements or “jumping genes” that can copy- and-paste their sequences to new locations in the host genome. The jumping ability, or retrotransposition efficiency, of individual L1 elements can be evaluated using a cultured cell assay in which the L1 is tagged in its 3’ untranslated region (3’UTR) with a reporter gene that becomes expressed upon successful retrotransposition. In a previous study, authors Han and Boeke reported that the retrotransposition efficiency of a mouse L1 element could be enhanced dramatically by synthetically increasing the GC content of the L1 open reading frames (ORFs) without changing their amino acid sequence. Curiously, a similarly constructed synthetic human L1 achieved only a modest increase in retrotransposition efficiency over the native element. Here, we find that two coding changes and a partial deletion comprising a guanine-rich region (GRR) of the mouse L1 3’UTR sequence which occurred during construction of the synthetic mouse L1 reporter are responsible for the increased jumping of the synthetic mouse L1 construct relative to the native L1spa element. We find that the presence/absence and also the placement of this GRR 3’UTR region within the reporter construct impact ORF1p expression and engineered L1 retrotransposition efficiency. Together, our study reconciles the disparate impacts of synthetic sequences upon human and mouse L1 retrotransposition efficiency, prompts a reconsideration of numerous studies using synthetic L1 constructs, and will inform the ongoing use of synthetic and natural mouse L1 reporter constructs in vivo and in vitro.