SEMPER: Stoichiometric expression of mRNA polycistrons by eukaryotic ribosomes for compact, ratio-tunable multi-gene expression

Creators: Duan, Mengtong; Dev, Ishaan; Lu, Andrew; Ayrapetyan, Goar; You, Mei Yi; Shapiro, Mikhail G.¹

1. California Institute of Technology

Abstract

Here, we present a method for expressing multiple open reading frames (ORFs) from single transcripts using the leaky scanning model of translation initiation. In this approach termed “stoichiometric expression of mRNA polycistrons by eukaryotic ribosomes” (SEMPER), adjacent ORFs are translated from a single mRNA at tunable ratios determined by their order in the sequence and the strength of their translation initiation sites. We validate this approach by expressing up to three fluorescent proteins from one plasmid in two different cell lines. We then use it to encode a stoichiometrically tuned polycistronic construct encoding gas vesicle acoustic reporter genes that enables efficient formation of the multi-protein complex while minimizing cellular toxicity. We also demonstrate that SEMPER enables polycistronic expression of recombinant monoclonal antibodies from plasmid DNA and of two fluorescent proteins from single mRNAs made through in vitro transcription. Finally, we provide a probabilistic model to elucidate the mechanisms underlying SEMPER. A record of this paper’s transparent peer review process is included in the supplemental information.

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Acknowledgement

The authors thank Michael Elowitz for helpful discussions, James Linton for equipment training, and Eman Elsheikh and Arul Goel for experimental support. This work was supported by the National Institutes of Health (R01EB018975 to M.G.S.), the Millard and Muriel Jacobs Genetics and Genomics Laboratory at the California Institute of Technology, and the Flow Cytometry and Cell Sorting Facility at the California Institute of Technology. Related research in the Shapiro laboratory is supported by the Chan Zuckerberg Initiative. M.G.S. is an investigator of the Howard Hughes Medical Institute. BioRender.com

Contributions

M.D., I.D., and M.G.S. conceived and executed the study, analyzed data, and wrote the manuscript. M.D. and M.G.S. developed the initial hypothesis. M.D. and I.D. constructed the fluorescent protein SEMPER breadboarding system. M.D. performed and analyzed gas vesicle expression and ultrasound experiments as well as antibody expression and secretion experiments. A.L. prepared the IVT mRNA protocol. I.D. developed the SEMPER IVT mRNA breadboarding system. I.D. performed and analyzed IVT mRNA experiments and 2-ORF and 3-ORF FP expression experiments and developed the computational model. G.A. assisted in plasmid design and construction. M.Y.Y. assisted in the development of methionine-less FPs. M.D. and A.L. performed apoptosis assays.

Data Availability

Document S1. Figures S1–S13, Tables S1–S8, and supplemental references.

Table S9. Genetic parts, primers, and plasmid sequences used to construct plasmids and IVT mRNA. Please refer to the attached Table S9 document.

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