Tuning Gene Expression by Hairpin Elements near the Start Codon of mRNA in Mammalian Cells
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Abstract: The hairpin element (HpE) near the start codon in the 5′ UTR was developed to tune the mRNA translation in mammalian cells. The parameters of HpEs including thermodynamic stability, GC content, and distance between HpEs and the 5′ cap were investigated. The parameters of HpEs including thermodynamic stability, GC content, and distance between HpEs and the 5′ cap were investigated, which influenced the mRNA expression level. In addition, the start codon and the upstream open reading frame sequestered within the structures of HpEs also reduced the translation initiation. In summary, this study shows that the simple engineering HpE structure can be efficiently adopted for gene expression regulation. The predictable controllability of this simple cloning strategy can potentially achieve precise gene expression regulation in different mammalian cell types.
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Key words:
- Gene expression regulation /
- Hairpin element /
- Start codon /
- mRNA
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Figure 1. (a) Schematic diagram of the reporter vector expressed in mammalian cells. The HpE is inserted upstream of the ORF of the reporter gene. (b) and (c) Influence of the HpEs in 5′ UTR of mRNAs in gene expression regulation. Normalized relative fluorescence levels are plotted in (b). The histograms EGFP mode fluorescence are present in (c). Error bar means standard deviation for three biological repetitions.
Figure 2. Stability of HpE in the 5′ UTR of mRNA tunes gene expression levels in HEK293T cells. (a) Impacts of HpEs with varied free energy in a constant 50% GC content; (b) influence of GC contents of HpEs in the constant free energy. Error bars mean standard deviation for three biological repetitions.
Figure 3. Distance between caps and HpEs influences the mRNA translation levels. (a) Illustration of mRNAs containing hairpins with various cap distances away from the cap. (b) Microscopy images of EGFP fluorescence of 37.5% GC content. Normalized gene expression levels of different cap distances: HpEs of the constant (c) 37.5% GC content, and (d) 53% GC content. Error bars mean standard deviation for three biological repetitions.
Figure 4. Start codon sequestered within HpEs tune gene expression levels in HEK293T cells. (a) Illustration of mRNAs containing hairpin structures at various stem lengths with the concealed start codon. (b) Relative normalized EGFP fluorescence levels of genes containing different HpEs. Error bars mean standard deviation for three biological repetitions.
Figure 5. Influence of uORF sequestered within the HpEs in translation control. (a) Illustration of mRNAs containing uORF and hairpin structures. (b) Relative normalized EGFP fluorescence levels of genes containing uORFs within different structures in the 5′ UTR of the gene. Error bars mean standard deviation for three biological repetitions.
Table I. Parameters of HpEs for mRNA translation control (cap distance: 14 nt).
HpEs Stability/(kcal/mol) Stem length/nt GC content/% H1 −47.2 21 76.2 H2 −40.0 18 77.8 H3 −34.6 16 75.0 H4 −39.5 21 61.9 H5 −32.3 18 61.1 H6 −34.4 17 70.6 
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Table II. Parameters of HpEs with AUG in the stem (cap distance: 6 nt).
HpEs Stability/ (kcal/mol) Stem length/nt GC content/% 16AUG –30.1 16 75 19AUG –36.1 19 68 22AUG −43.0 22 68 25AUG −48.7 25 68 28ATG −57.0 28 75
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