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Tracking endogenous proteins based on RNA editing-mediated genetic code expansion

Nature Chemical Biology (2024)Cite this article

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Abstract

Protein labeling approaches are important to study proteins in living cells, and genome editing tools make it possible to tag endogenous proteins to address the concerns associated with overexpression. Here we established RNA editing-mediated noncanonical amino acids (ncAAs) protein tagging (RENAPT) to site-specifically label endogenous proteins with ncAAs in living cells. RENAPT labels protein in a temporary and nonheritable manner and is not restricted by protospacer adjacent motif sequence. Using a fluorescent ncAA or ncAA with a bio-orthogonal reaction handle for subsequent dye labeling, we demonstrated that a variety of endogenous proteins can be imaged at their specific subcellular locations. In addition, two proteins can be tagged individually and simultaneously using two different ncAAs. Furthermore, endogenous ion channels and neuron-specific proteins can be real-time labeled in primary neurons. Thus, RENAPT presents a promising platform with broad applicability for tagging endogenous proteins in living cells to study their localization and functions.

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Fig. 1: Graphical representation showing the principle of RENAPT and evaluation of RNA editing efficiency.
Fig. 2: Site-specific post-transcriptional incorporation of ncAAs into proteins encoded by endogenous genes using RENAPT.
Fig. 3: Subcellular localization of different endogenous proteins in U2OS cells by RENAPT.
Fig. 4: Dual labeling of target proteins by RENAPT.
Fig. 5: Applications of RENAPT in super-resolution imaging.
Fig. 6: Labeling of endogenous proteins in primary mouse hippocampal neurons by RENAPT.

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The authors declare that the main data supporting the study are provided within this article and its Supplementary Information. Source data are provided with this paper.

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Acknowledgements

This work was financially supported by the National Key Research and Development Program of China (2022YFA0912400), the National Natural Science Foundation of China (92253301, 22325701, U22A20332, 92156025 and 82003681), the National Science and Technology Major Project Program (2021YFA0909900 and 2022YFC3400600), China Postdoctoral Science Foundation (2020M680255) and the Beijing Natural Science Foundation (JQ20034). We acknowledge X. Zhang for assistance with high-resolution protein mass spectrometry and X. Yuan and Z. Jiang for assisting on confocal and FACS at the State Key Laboratory of Natural and Biomimetic Drugs. We acknowledge T. Chi from the ShanghaiTech University School of Life Sciences and Technology for kindly providing the CURE system and Split GFP reporter system and H. Yang from the Institute of Neuroscience for kindly providing the Cas13X RNA editor. We thank Guangzhou Computational Super-resolution Biotech for live-cell imaging by using their commercial super-resolution microscope (HIS-SIM), data acquisition, SR image reconstruction, analysis and discussion.

Author information

Author notes

  1. Xinyu Ling

    Present address: Department of Genetics, Yale University School of Medicine, New Haven, CT, USA

  2. These authors contributed equally: Min Hao, Xinyu Ling, Yi Sun.

Authors and Affiliations

  1. State Key Laboratory of Natural and Biomimetic Drugs, Chemical Biology Center, Department of Molecular and Cellular Pharmacology, Pharmaceutical Sciences, Peking University, Beijing, China

    Min Hao, Xinyu Ling, Yi Sun, Xue Wang, Wenzhe Li, Liying Chang, Zhiying Zeng, Xiaomeng Shi & Tao Liu

  2. State Key Laboratory of Membrane Biology, Institute of Molecular Medicine, Peking-Tsinghua Center for Life Sciences, College of Future Technology, Peking University, Beijing, China

    Mengxiao Niu & Liangyi Chen

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Contributions

M.H., X.L. and T.L. designed the experiments and revised the manuscript. M.H. wrote the manuscript and performed most of the experiments. M.H. performed confocal imaging and super-resolution imaging and analyzed data. Y.S. performed primary mouse hippocampal neuron culture. M.H. and X.W. performed plasmid construction, RNA-seq analysis, FACS and western blotting. W.L. assisted in confocal imaging. L.C. assisted with plasmid construction. Z.Z. performed chemical synthesis. X.S. assisted with mass spectrometry data analysis. M.N. assisted with marker protein selection. L.C. assisted in checking all the images. All authors approved the final manuscript.

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Correspondence to Tao Liu.

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Extended data

Extended Data Fig. 1 Efficiency of RNA editing.

a. The split GFP reporter system for evaluating RNA editing efficiency. The reconstituted GFP can be detected by FACS. b. Representatives of FACS plot chromatograph. The gating was initially performed based on FSC/SSC for live cells. Subsequently, a gate was applied to distinguish between positive and negative cells using control cells (transfected with scramble RNA) that were treated in the same manner on the day of each experiment. c. Schematics of nonsense-mediated mRNA decay (NMD) knockdown for enhanced RNA editing. NMD is a mechanism that degrades aberrant mRNAs containing premature nonsense codons in eukaryotes. d. SiRNA-mediated knockdown of Upf1 or SMG-1 results in a reduction in the expression of Upf1 or SMG-1 mRNA. The relative gene expression was calculated using the threshold cycle (2-ΔΔCT) method with the GAPDH mRNA as internal control. Data are presented as the mean ± SD, n = 3 independent samples. Source data are provided as a source data file. The primers are listed in Supplementary Table 1.

Source data

Extended Data Fig. 2 Transfection efficiency and editing efficiency of different sites in endogenous proteins.

a. Transfection efficiency of different sites in GRP94. The gating was initially performed based on FSC/SSC for live cells. Subsequently, a gate was applied to distinguish between positive and negative cells using control cells (transfected with scramble RNA) that were treated in the same manner on the day of each experiment. b. Editing efficiency of different sites in GRP94. c. Percentage of labeling efficiency in transfected cells. d. Western blotting analysis of stopped versus incorporates ncAA. e. Western blot analysis was performed for GRP94. HEK293 cells were transfected with plasmids (MbPylRS-AF/tRNACUA, RESCUE-S, gRNA) and grown with 250 μM TCO-L-lysine for 48 hours. The TCO-L-lysine medium was removed and the cells were washed twice with fresh medium. Then, the cells were incubated with 10 μM tetrazine dye diluted in fresh medium for 10 min at 37 °C with 5% CO2. After that, the cells were washed twice with fresh medium. The cells were lysed and centrifuged at 15000g for 30 minutes to clear the lysate. GRP94 antibody was labeled with AF488, and β-tubulin was labeled with Cy3. The fluorescence signals were visualized using Typhoon fluorescence imaging system. Lane A represents the wild-type (WT) sample, while lane B represents the PENAPT labeled sample. A protein marker (SMOBIO Technology, cat. no. PM2511) was used for size reference. Data are presented as the mean ± SD, n = 3 independent samples. Scale bar: 100 pixels. Source data are provided as a source data file.

Source data

Extended Data Fig. 3 Label of different endogenous proteins in HEK293 cells by RENAPT.

a. The mutations introduced at the post-translational (mRNA) level include GRP94Q452UAG, STX6Q212UAG, ATP1A1Q383UAG, ATP5AQ439UAG, IGF2-RQ506UAG and H3.3AQ56UAG and the corresponding proteins are localized to the endoplasmic reticulum, Golgi, cell membrane, mitochondria, lysosomes and nucleus, respectively. b. RNA editing efficiency on different sites of different protein coding mRNA. Cells were stained with Mito-Tracker Red CMXRos, Lyso-Tracker Red, ER-Tracker Red, Cell Plasma Membrane Staining Kit with DiI, Golgi-Tracker Red and Hoechst 33342, respectively. Scale bar: 20 μm. Data are presented as the mean ± SD, n = 3 independent samples. Source data are provided as a source data file.

Source data

Extended Data Fig. 4 Optimization of reaction conditions.

a. The optimal RS for introducing TCO-K. b. Concentration optimization of SiR-Tz dye. The endogenous protein ATP5AQ439UAG was used for optimization of SiR-Tz dye concentration. Scale bar: 20 μm. c. Cytotoxicity of different ncAA concentrations; d. Cytotoxicity of different plasmids transfection. The cytotoxicity of RENAPT was analyzed using a CCK-8. HEK293 cells were seeded into 96-well plates at a density of 2 × 103 cells per well. The cells were collected at 48 h transfection and cultured with 10 μL CCK-8 reagent. After incubating for 2 h at 37 °C, the absorbance at 450 nm was tested using a Microplate Reader. Source data are provided as a source data file. Data are presented as the mean ± SD, n = 3 independent samples.

Source data

Extended Data Fig. 5 Validation of selectivity and specificity of RENAPT system.

a. Subcellular localization of endogenous proteins in HEK293 cells visualized using the SiR-Tz dye. The mutations introduced at the post-translational (mRNA) level include ATP5AQ439UAG and IGF2-RQ506UAG, and the corresponding proteins are localized to the mitochondria and lysosome. Scale bar: 20 μm. b. Validation of selectivity and specificity of RENAPT system. The selectivity and specificity of RENAPT system were verified by labeling endogenous ATP5A protein in U2OS cells with different conditions. To illustrate the co-localization, the nucleus and mitochondria were stained by Hoechst 33342 and Mitochondrial Tracker-Red. Scale bar: 5 μm.

Extended Data Fig. 6 Labeling of ATP5A and GRP94 by RENAPT and antibodies.

The cells were immunostained with anti-ATP5A or anti-GRP94 primary antibody, followed by CY3-conjugated secondary antibody. Scale bar: 5 μm.

Extended Data Fig. 7 Subcellular localization of different endogenous proteins by RENAPT.

The mutations introduced at the post-transcriptional (mRNA) level include STX6Q212UAG, H3.3AQ56UAG and RAB7AQ27UAG, and the corresponding proteins are localized to the Golgi, nucleus and cell membrane, respectively. The cells were stained with Golgi-Tracker Red, Hoechst 33342 and Cell Plasma Membrane Staining Kit with DiI, respectively. Scale bar: 5 μm.

Extended Data Fig. 8 Pearson correlation coefficient (PCC) was used for quantitative analysis of colocalization.

a. Endogenous protein PCC coefficient in U2OS cells. b. PCC coefficient for dual-labeling of histone H3.3a-mCherry and histone H3.3b-eGFP. c. PCC coefficient for dual-labeling of endogenous ATP5A and IGF2-R. d. PCC plot representing the colocalization between target protein and tracker. Data are presented as the mean ± SD, n = 3 independent samples. Source data are provided as a source data file.

Source data

Extended Data Fig. 9 RENAPT method for labeling different endogenous proteins in primary hippocampal neurons.

a. Labeling different endogenous proteins in primary hippocampal neurons. 4 days after plating, primary mouse hippocampal neurons were transfected with RESCUE-S-mCherry, gRNA and NES PylRS/tRNACUAPyl. After 2 days of incubation with Anap, neurons were imaged on the confocal scanning microscope. Scale bar: 20 μm. b. The specificity of genetic incorporation was confirmed by the control group (scramble gRNA), where no fluorescence signal of SiR-Tz was detected. 8 days after plating, primary mouse hippocampal neurons were transfected with RESCUE-S, gRNA and NES PylRS/tRNACUAPyl. After 3 days of incubation with TCO-K, the neurons were labeled with the dye (SiR-Tz) and subsequently imaged using a confocal scanning microscope with a 100× oil objective. Scale bar: 5 μm. c. Distribution of Nav1.6 in primary mouse hippocampal neurons transfected with eGFP and anti-Nav1.6 immunostaining. The neurons were transfected with RESCUE-S-eGFP, gRNA and NES PylRS/tRNACUAPyl. After 3 days of incubation with TCO-K, the neurons were labeled with the dye (SiR-Tz). Then, the neurons were labeled with Nav1.6 antibody and imaged on a confocal scanning microscope. Scale bar: 5 μm.

Extended Data Fig. 10 Relevant components and sizes of the plasmid.

Plasmid 1 represents the RNA editing system, comprising primarily dPspCas13b, ADAR2dd, and gRNA. Plasmid 2 contains the essential elements of the non-natural amino acid insertion system, including orthogonal TCOK-RS and tRNA.

Supplementary information

Supplementary Information

Supplementary Tables 1–3.

Reporting Summary

Supplementary Video 1

Mitochondrial Tracker Red.

Supplementary Video 2

Mitochondrial protein ATP5A.

Supplementary Video 3

Colocalization of ATP5A and Tracker.

Supplementary Video 4

Lysosome Tracker Red.

Supplementary Video 5

Lysosome protein IGF2-R.

Supplementary Video 6

Colocalization of IGF2-R and Tracker.

Source data

Source Data Fig. 1

Statistical source data.

Source Data Fig. 2

Statistical source data.

Source Data Extended Data Fig. 1

Statistical source data.

Source Data Extended Data Fig. 2

Statistical source data of Extended Data Fig. 2a.

Source Data Extended Data Fig. 2

Unprocessed western blots of Extended Data Fig. 2d,e.

Source Data Extended Data Fig. 3

Statistical source data.

Source Data Extended Data Fig. 4

Statistical source data.

Source Data Extended Data Fig. 8

Statistical source data.

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Hao, M., Ling, X., Sun, Y. et al. Tracking endogenous proteins based on RNA editing-mediated genetic code expansion. Nat Chem Biol (2024). https://doi.org/10.1038/s41589-023-01533-w

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