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Structures in the 5′ untranslated regions (UTRs) of mRNAs can physically modulate translation efficiency by impeding the scanning ribosome or by sequestering the translational start site. We assessed the impact of stable protein binding in 5′- and 3′-UTRs on translation efficiency by targeting the MS2 coat protein to a reporter RNA via its hairpin recognition site. Translation was assessed from the reporter RNA when coexpressed with MS2 coat proteins of varying affinities for the RNA, and at different expression levels. Binding of high-affinity proteins in the 5′-UTR hindered translation, whereas no effect was observed when the coat protein was targeted to the 3′-UTR. Inhibition of translation increased with coat protein concentration and affinity, reaching a maximum of 50%–70%. MS2 proteins engineered to bind two reporter mRNA sites had a stronger effect than those binding a single site. Our findings demonstrate that protein binding in an mRNA 5′-UTR physically impedes translation, with the effect governed by affinity, concentration, and sterics.
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This content will become publicly available on June 1, 2026
All Probes Plasmids (APPs) for multicolor and long-term tracking of single-mRNA translation dynamics
Live-cell single-mRNA imaging of translation is inherently challenging, demanding precise optimization of multiple imaging components. To simplify these experiments, we developed All Probes Plasmids (APPs)—a panel of plasmids encoding all the necessary probes for imaging at optimized relative expression levels. APPs incorporate widely used translation tags, fluorescent proteins, and mRNA labeling systems, streamlining both multiplexed imaging and reporter immobilization. By cotransfecting just two plasmids—a reporter and an APP—individual translation sites can be visualized in living cells with high signal-to-noise. We demonstrate how APPs facilitate high-fidelity multicolor translation imaging, long-term single-mRNA tracking, and fluorescence correlation spectroscopy to quantify ribosome kinetics. By lowering technical barriers and enhancing experimental flexibility, APPs provide a versatile platform for advancing single-mRNA translation research in living cells.
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- Award ID(s):
- 1845761
- PAR ID:
- 10611619
- Editor(s):
- Liao, Ya-Cheng
- Publisher / Repository:
- Molecular Biology of the Cell
- Date Published:
- Journal Name:
- Molecular Biology of the Cell
- Volume:
- 36
- Issue:
- 6
- ISSN:
- 1059-1524
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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