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Development of a split fluorescent protein-based RNA live-cell imaging system to visualize mRNA distribution in plants
BACKGROUND: RNA live-cell imaging systems have been used to visualize subcellular mRNA distribution in living cells. The RNA-binding protein (RBP)-based RNA imaging system exploits specific RBP and the corresponding RNA recognition sequences to indirectly label mRNAs. Co-expression of fluorescent pr...
Autores principales: | , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
BioMed Central
2022
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8822845/ https://www.ncbi.nlm.nih.gov/pubmed/35130941 http://dx.doi.org/10.1186/s13007-022-00849-3 |
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author | Huang, Nien-Chen Luo, Kai-Ren Yu, Tien-Shin |
author_facet | Huang, Nien-Chen Luo, Kai-Ren Yu, Tien-Shin |
author_sort | Huang, Nien-Chen |
collection | PubMed |
description | BACKGROUND: RNA live-cell imaging systems have been used to visualize subcellular mRNA distribution in living cells. The RNA-binding protein (RBP)-based RNA imaging system exploits specific RBP and the corresponding RNA recognition sequences to indirectly label mRNAs. Co-expression of fluorescent protein-fused RBP and target mRNA conjugated with corresponding RNA recognition sequences allows for visualizing mRNAs by confocal microscopy. To minimize the background fluorescence in the cytosol, the nuclear localization sequence has been used to sequester the RBP not bound to mRNA in the nucleus. However, strong fluorescence in the nucleus may limit the visualization of nucleus-localized RNA and sometimes may interfere in detecting fluorescence signals in the cytosol, especially in cells with low signal-to-noise ratio. RESULTS: We eliminated the background fluorescence in the nucleus by using the split fluorescent protein-based approach. We fused two different RBPs with the N- or C-terminus of split fluorescent proteins (FPs). Co-expression of RBPs with the target mRNA conjugated with the corresponding RNA recognition sequences can bring split FPs together to reconstitute functional FPs for visualizing target mRNAs. We optimized the system with minimal background fluorescence and used the imaging system to visualize mRNAs in living plant cells. CONCLUSIONS: We established a background-free RNA live-cell imaging system that provides a platform to visualize subcellular mRNA distribution in living plant cells. |
format | Online Article Text |
id | pubmed-8822845 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2022 |
publisher | BioMed Central |
record_format | MEDLINE/PubMed |
spelling | pubmed-88228452022-02-08 Development of a split fluorescent protein-based RNA live-cell imaging system to visualize mRNA distribution in plants Huang, Nien-Chen Luo, Kai-Ren Yu, Tien-Shin Plant Methods Methodology BACKGROUND: RNA live-cell imaging systems have been used to visualize subcellular mRNA distribution in living cells. The RNA-binding protein (RBP)-based RNA imaging system exploits specific RBP and the corresponding RNA recognition sequences to indirectly label mRNAs. Co-expression of fluorescent protein-fused RBP and target mRNA conjugated with corresponding RNA recognition sequences allows for visualizing mRNAs by confocal microscopy. To minimize the background fluorescence in the cytosol, the nuclear localization sequence has been used to sequester the RBP not bound to mRNA in the nucleus. However, strong fluorescence in the nucleus may limit the visualization of nucleus-localized RNA and sometimes may interfere in detecting fluorescence signals in the cytosol, especially in cells with low signal-to-noise ratio. RESULTS: We eliminated the background fluorescence in the nucleus by using the split fluorescent protein-based approach. We fused two different RBPs with the N- or C-terminus of split fluorescent proteins (FPs). Co-expression of RBPs with the target mRNA conjugated with the corresponding RNA recognition sequences can bring split FPs together to reconstitute functional FPs for visualizing target mRNAs. We optimized the system with minimal background fluorescence and used the imaging system to visualize mRNAs in living plant cells. CONCLUSIONS: We established a background-free RNA live-cell imaging system that provides a platform to visualize subcellular mRNA distribution in living plant cells. BioMed Central 2022-02-08 /pmc/articles/PMC8822845/ /pubmed/35130941 http://dx.doi.org/10.1186/s13007-022-00849-3 Text en © The Author(s) 2022 https://creativecommons.org/licenses/by/4.0/Open AccessThis 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 licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence 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 licence, visit http://creativecommons.org/licenses/by/4.0/ (https://creativecommons.org/licenses/by/4.0/) . The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/ (https://creativecommons.org/publicdomain/zero/1.0/) ) applies to the data made available in this article, unless otherwise stated in a credit line to the data. |
spellingShingle | Methodology Huang, Nien-Chen Luo, Kai-Ren Yu, Tien-Shin Development of a split fluorescent protein-based RNA live-cell imaging system to visualize mRNA distribution in plants |
title | Development of a split fluorescent protein-based RNA live-cell imaging system to visualize mRNA distribution in plants |
title_full | Development of a split fluorescent protein-based RNA live-cell imaging system to visualize mRNA distribution in plants |
title_fullStr | Development of a split fluorescent protein-based RNA live-cell imaging system to visualize mRNA distribution in plants |
title_full_unstemmed | Development of a split fluorescent protein-based RNA live-cell imaging system to visualize mRNA distribution in plants |
title_short | Development of a split fluorescent protein-based RNA live-cell imaging system to visualize mRNA distribution in plants |
title_sort | development of a split fluorescent protein-based rna live-cell imaging system to visualize mrna distribution in plants |
topic | Methodology |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8822845/ https://www.ncbi.nlm.nih.gov/pubmed/35130941 http://dx.doi.org/10.1186/s13007-022-00849-3 |
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