Revisiting the impact of synthetic ORF sequences on engineered LINE-1 retrotransposition
5 affiliations
- 1Mater Research Institute - University of Queensland, Woolloongabba, Queensland, Australia.
- 2Department of Biochemistry and Molecular Biology, Tulane University School of Medicine, New Orleans, Louisiana, United States of America.
- 3Institute for Systems Genetics and Department of Biochemistry and Molecular Pharmacology, NYU Langone Health, New York, New York, United States of America.
- 4Department of Biomedical Engineering, NYU Tandon School of Engineering, Brooklyn, New York, United States of America.
- 5Queensland Brain Institute, University of Queensland, Brisbane, Queensland, Australia.
Abstract
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. 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. Our study reveals a potential role for the GRR sequence in L1 protein expression and will inform the ongoing use of synthetic and natural mouse L1 reporter constructs in vivo and in vitro.
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BibTeX
@article{chan2026revisiting,
title = {{Revisiting the impact of synthetic ORF sequences on engineered LINE-1 retrotransposition}},
author = {Dorothy Chan and Stephanie Workman and Nathan Smits and Alexander L Carleton and Patricia Gerdes and Jeffrey S Han and Jef D Boeke and Geoffrey J Faulkner and Sandra R Richardson},
journal = {PLOS Genetics},
year = {2026},
month = oct,
doi = {10.1371/journal.pgen.1012340},
url = {https://journals.plos.org/plosgenetics/article?id=10.1371/journal.pgen.1012340},
note = {CC BY},
}
RIS
TY - JOUR
TI - Revisiting the impact of synthetic ORF sequences on engineered LINE-1 retrotransposition
AU - Dorothy Chan
AU - Stephanie Workman
AU - Nathan Smits
AU - Alexander L Carleton
AU - Patricia Gerdes
AU - Jeffrey S Han
AU - Jef D Boeke
AU - Geoffrey J Faulkner
AU - Sandra R Richardson
PY - 2026
DA - 2026/10/16
JO - PLOS Genetics
DO - 10.1371/journal.pgen.1012340
UR - https://journals.plos.org/plosgenetics/article?id=10.1371/journal.pgen.1012340
AB - 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. 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. Our study reveals a potential role for the GRR sequence in L1 protein expression and will inform the ongoing use of synthetic and natural mouse L1 reporter constructs in vivo and in vitro.
SN - 1553-7404
SN - 1553-7390
ER -