Tuning gene expression with synthetic upstream open reading frames
Open accessAbstract
We engineered short ORFs and used them to control the expression level of recombinant proteins. These short ORFs, encoding a two-amino acid peptide, were placed upstream of an ORF encoding a protein of interest. Insertion of these upstream ORFs (uORFs) resulted in suppression of protein expression. By varying the base sequence preceding the uORF, we sought to vary the translation initiation rate of the uORF and subsequently control the degree of this suppression. Using this strategy, we generated a library of RNA sequence elements that can specify protein expression over a broad range of levels. By also using multiple uORFs in series and non-AUG start codons, we were able to generate particularly low expression levels, allowing us to achieve expression levels spanning three orders of magnitude. Modeling supported a mechanism where uORFs shunt the flow of ribosomes away from the downstream protein-coding ORF. With a lower translation initiation rate at the uORF, more ribosomes “leak” past the uORF; consequently, more ribosomes are able to reach and translate the downstream ORF. We report expression control by engineering uORFs and translation initiation to be robust, predictable, and reproducible across all cell types tested. We propose control of translation initiation as a primary method of choice for tuning expression in mammalian systems.
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A fully rational approach to designing functional 5′ UTRs has not yet been implemented.
“While there has been some success in this effort in prokaryotes, yeast and even mammalian cells27,34–36, a fully rational approach to designing functional 5′ UTRs has not yet been implemented.”
From Human 5′ UTR design and variant effect prediction from a massively parallel translation assay (2019) · cites this paper as [27]
The opening discussion says there were few systematic approaches for precisely controlling recombinant-protein translation in mammalian cells, which supports the claim’s general concern about design methods. It does not say that a fully rational approach to designing functional 5′ UTRs had never been implemented; the Discussion instead describes the authors’ own systematic approach using engineered RNA leaders. The claim is broader than the source supports. (LLM, conf=0.80)
Experimental — accuracy not yet measuredOptimus 5-Prime MRL predictions correlated well with independently reported fluorescence levels for 77 5′ UTRs previously designed by Ferreira et al. (r2: 0.73-0.85).
“Our model’s MRL predictions correlated well with the independently reported fluorescence levels (r2: 0.73–0.85, Supplementary Fig. 9).”
From Human 5′ UTR design and variant effect prediction from a massively parallel translation assay (2019) · cites this paper as [27]
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p21 in cancer: Intricate networks and multiple activities
Abbas, T., & Dutta, A. (2009) · Nature Reviews Cancer · doi:10.1038/nrc2657 · Cited by 2,712 · see citing works
Flagged first, then by citations
Research works printed but not linked: 26
- Salis HM, Mirsky EA, Voigt CA (2009) Automated design of synthetic ribosome binding sites to control protein expression. Nat Biotechnol 27(10):946-950.
- Hinnebusch AG (2011) Molecular mechanism of scanning and start codon selection in eukaryotes. Microbiol Mol Biol Rev 75(3):434-467.
- Jackson RJ, Hellen CU, Pestova TV (2010) The mechanism of eukaryotic translation initiation and principles of its regulation. Nat Rev Mol Cell Biol 11(2):113-127.
- Kozak M (1981) Possible role of flanking nucleotides in recognition of the AUG ini- tiator codon by eukaryotic ribosomes. Nucleic Acids Res 9(20):5233-5252.
- Kozak M (1984) Compilation and analysis of sequences upstream from the trans- lational start site in eukaryotic mRNAs. Nucleic Acids Res 12(2):857-872.
- Kozak M (1986) Point mutations define a sequence flanking the AUG initiator codon that modulates translation by eukaryotic ribosomes. Cell 44(2):283-292.
- Calvo SE, Pagliarini DJ, Mootha VK (2009) Upstream open reading frames cause widespread reduction of protein expression and are polymorphic among humans. Proc Natl Acad Sci USA 106(18):7507-7512.
- Racine T, Duncan R (2010) Facilitated leaky scanning and atypical ribosome shunting direct downstream translation initiation on the tricistronic S1 mRNA of avian reovirus. Nucleic Acids Res 38(20):7260-7272.
- Stacey SN, et al. (2000) Leaky scanning is the predominant mechanism for translation of human papillomavirus type 16 E7 oncoprotein from E6/E7 bicistronic mRNA. J Virol 74(16):7284-7297.
- Kozak M (2002) Emerging links between initiation of translation and human diseases. Mamm Genome 13(8):401-410.
- el-Deiry WS, et al. (1993) WAF1, a potential mediator of p53 tumor suppression. Cell 75(4):817-825.
- Harper JW, Adami GR, Wei N, Keyomarsi K, Elledge SJ (1993) The p21 Cdk-interacting protein Cip1 is a potent inhibitor of G1 cyclin-dependent kinases. Cell 75(4):805-816.
- Fussenegger M, Schlatter S, Dätwyler D, Mazur X, Bailey JE (1998) Controlled pro- liferation by multigene metabolic engineering enhances the productivity of Chinese hamster ovary cells. Nat Biotechnol 16(5):468-472.
- Kozak M (1984) Selection of initiation sites by eucaryotic ribosomes: Effect of in- serting AUG triplets upstream from the coding sequence for preproinsulin. Nucleic Acids Res 12(9):3873-3893.
- Kozak M (1987) Effects of intercistronic length on the efficiency of reinitiation by eucaryotic ribosomes. Mol Cell Biol 7(10):3438-3445.
- Kozak M (1989) Circumstances and mechanisms of inhibition of translation by sec- ondary structure in eucaryotic mRNAs. Mol Cell Biol 9(11):5134-5142.
- Babendure JR, Babendure JL, Ding JH, Tsien RY (2006) Control of mammalian trans- lation by mRNA structure near caps. RNA 12(5):851-861.
- Kozak M (1990) Downstream secondary structure facilitates recognition of initiator codons by eukaryotic ribosomes. Proc Natl Acad Sci USA 87(21):8301-8305.
- Ferreira JP, Peacock RWS, Lawhorn IEB, Wang CL (2011) Modulating ectopic gene expression levels by using retroviral vectors equipped with synthetic promoters. Syst Synth Biol 5(3-4):131-138.
- Ferreira JP, Lawhorn IE, Peacock RW, Wang CL (2012) Quantitative assessment of Ras over-expression via shotgun deployment of vectors utilizing synthetic promoters. Integr Biol (Camb) 4(1):108-114.
- Peacock RW, Lawhorn IE, Ferreira JP, Wang CL (2012) Flow cytometry of v-Abl transformed pre-B cells heterogeneous in ectopic expression levels reveals Ras dose- response. J Immunol Methods 384(1-2):177-183.
- Jensen PR, Hammer K (1998) Artificial promoters for metabolic optimization. Bio- technol Bioeng 58(2-3):191-195.
- Peacock RW, Wang CL (2011) A genetic reporter system to gauge cell proliferation rate. Biotechnol Bioeng 108(9):2003-2010.
- Wang CL, Harper RA, Wabl M (2004) Genome-wide somatic hypermutation. Proc Natl Acad Sci USA 101(19):7352-7356.
- Naviaux RK, Costanzi E, Haas M, Verma IM (1996) The pCL vector system: Rapid pro- duction of helper-free, high-titer, recombinant retroviruses. J Virol 70(8):5701-5705.
- Hofacker IL (2004) RNA secondary structure analysis using the Vienna RNA package. Curr Protoc Bioinformatics Chap 12:Unit 12.2, 10.1002/0471250953.bi1202s04. · DOI
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Functional 5′ UTR mRNA structures in eukaryotic translation regulation and how to find them
Leppek, K., Das, R., & Barna, M. (2018) · Nature Reviews Molecular Cell Biology · doi:10.1038/nrm.2017.103 · Cited by 949 · see citing works
Human 5′ UTR design and variant effect prediction from a massively parallel translation assay
Sample, P. J., Wang, B., Reid, D. W., Presnyak, V., McFadyen, I. J., Morris, D. R., & Seelig, G. (2019) · Nature Biotechnology · doi:10.1038/s41587-019-0164-5 · Cited by 442 · see citing works
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Deverman, B. E., Ravina, B. M., Bankiewicz, K. S., Paul, S. M., & Sah, D. W. Y. (2018) · Nature Reviews Drug Discovery · doi:10.1038/nrd.2018.110 · Cited by 306 · see citing works
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A live-cell marker to visualize the dynamics of stable microtubules throughout the cell cycle
Jansen, K. I., Iwanski, M. K., Burute, M., & Kapitein, L. C. (2023) · Journal of Cell Biology · doi:10.1083/jcb.202106105 · Cited by 39 · see citing works
- Authors
- Ferreira, J. P., Overton, K. W., & Wang, C. L.
- Affiliations
- Stanford University (US)
- ORCID
- Overton, K. Wesley
- Venue
- Proceedings of the National Academy of Sciences
- Publisher
- National Academy of Sciences
- Published
- 2013-06-24
- Volume
- 110
- Issue
- 28
- Pages
- 11284-11289
- ISSN
- 0027-8424 (ISSN-L)
- 1091-6490
- PMC
- PMC3710870
- OpenAlex
- W1997465134
- Cited by
- 116
- References
- 1
- Citations by year
- 2026: 7 · 2025: 6 · 2024: 12 · 2023: 11 · 2022: 8 · 2021: 4 · 2020: 11 · 2019: 10 · 2018: 11 · 2017: 11 · 2016: 6 · 2015: 9 · 2014: 9 · 2013: 1
- FWCI
- 3.14
- Type
- journal article
- Language
- en
- Open access
- bronze
- doi.org
- Locations
- Proceedings of the National Academy of Sciences (publishedVersion)
- Europe PMC (PubMed Central) (publishedVersion)
- PubMed Central (publishedVersion)
- PubMed (publishedVersion)
- Funders
- National Science Foundation (CAREER, 0846392)
- Ellison Medical Foundation (AG-NS-0550-09)
- Johns Hopkins University
- University of California, San Francisco
- Topics
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › RNA and protein synthesis mechanisms
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Gene Regulatory Network Analysis
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Microbial Metabolic Engineering and Bioproduction
- Keywords
- upstream open reading frames, translation initiation, gene expression regulation, synthetic gene design, non-AUG start codons, mammalian cell expression systems
- MeSH terms
- Animals, Cell Line, Humans, Models, Genetic, Gene Expression, Open Reading Frames
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