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. 2020 Mar 10;11:1297. doi: 10.1038/s41467-020-15061-x

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© The Author(s) 2020

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Fig. 8 — HeLa cells were co-transfected with hmAG1 mRNAs containing an MS2-binding motif (cap analog: A-cap, modified nucleosides: N1mΨ or Ψ/5mC) and mRNAs that express tagRFP, DmrC-VPg(FCV), and MS2CP-1xDmrA or MS2CP(V29I)-1xDmrA. Then, A/C heterodimerizer was added. a Schematic diagram of drug-regulatable CaVT, which is composed of MS2CP-1xDmrA and DmrC-VPg(FCV). MS2CP-1xDmrA binds to the MS2 binding motif of target mRNAs. In the absence of A/C heterodimerizer, DmrC-VPg does not bind to DmrA, and there is no VPg-mediated translational activation. After the addition of A/C heterodimerizer, DmrA-DmrC interaction tethers VPg to the target mRNAs, and VPg activates translation. b, c Effects of modified nucleosides, sites, and variants of the MS2-binding motif and variants of MS2CP on translation. The fluorescence was measured by flow cytometry and means of the hmAG1/tagRFP ratio were normalized by the ratio in the reporter mRNA only (b) or 0 nM A/C heterodimerizer samples (c). The bar graph shows the average of three independent experiments (mean ± SD). Source data are provided as a Source Data file. d, e Dose-dependency of drug-regulatable CaVT. 1xMS2(U)site1-hmAG1 (cap analog: A-cap, modified nucleosides: N1mΨ), tagRFP, DmrC-VPg(FCV), and MS2CP-1xDmrA mRNAs were used for the transfection. Means of the hmAG1/tagRFP ratio were normalized by the ratio in 0 nM A/C heterodimerizer samples. The bar graph shows the average of three independent experiments (mean ± SD) (d). A representative histogram (e). Source data are provided as a Source Data file. f Time lapse images of drug-induced translational activation. 1xMS2(U)site1-hmAG1 (cap analog: A-cap, modified nucleosides: N1mΨ), DmrC-VPg(FCV), and MS2CP-1xDmrA mRNAs were used for the transfection. Each image was captured at the indicated time points after the addition of A/C heterodimerizer. Three independent experiments were performed to check reproducibility, and representative images are shown. The scale bar represents 200 μm.