Identification of RNA-Binding Protein Targets with HyperTRIBE in <i>Saccharomyces cerevisiae</i>
As a master regulator in cells, RNA-binding protein (RBP) plays critical roles in organismal development, metabolism and various diseases. It regulates gene expression at various levels mostly by specific recognition of target RNA. The traditional CLIP-seq method to detect transcriptome-wide RNA tar...
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| Format: | Article |
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MDPI AG
2023-05-01
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| Series: | International Journal of Molecular Sciences |
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| Online Access: | https://www.mdpi.com/1422-0067/24/10/9033 |
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| author | Weilan Piao Chong Li Pengkun Sun Miaomiao Yang Yansong Ding Wei Song Yunxiao Jia Liqun Yu Yanming Lu Hua Jin |
| author_facet | Weilan Piao Chong Li Pengkun Sun Miaomiao Yang Yansong Ding Wei Song Yunxiao Jia Liqun Yu Yanming Lu Hua Jin |
| author_sort | Weilan Piao |
| collection | DOAJ |
| description | As a master regulator in cells, RNA-binding protein (RBP) plays critical roles in organismal development, metabolism and various diseases. It regulates gene expression at various levels mostly by specific recognition of target RNA. The traditional CLIP-seq method to detect transcriptome-wide RNA targets of RBP is less efficient in yeast due to the low UV transmissivity of their cell walls. Here, we established an efficient HyperTRIBE (Targets of RNA-binding proteins Identified By Editing) in yeast, by fusing an RBP to the hyper-active catalytic domain of human RNA editing enzyme ADAR2 and expressing the fusion protein in yeast cells. The target transcripts of RBP were marked with new RNA editing events and identified by high-throughput sequencing. We successfully applied HyperTRIBE to identifying the RNA targets of two yeast RBPs, KHD1 and BFR1. The antibody-free HyperTRIBE has competitive advantages including a low background, high sensitivity and reproducibility, as well as a simple library preparation procedure, providing a reliable strategy for RBP target identification in <i>Saccharomyces cerevisiae</i>. |
| first_indexed | 2024-03-11T03:38:24Z |
| format | Article |
| id | doaj.art-47419248b4e7453badf772f28ed404bf |
| institution | Directory Open Access Journal |
| issn | 1661-6596 1422-0067 |
| language | English |
| last_indexed | 2024-03-11T03:38:24Z |
| publishDate | 2023-05-01 |
| publisher | MDPI AG |
| record_format | Article |
| series | International Journal of Molecular Sciences |
| spelling | doaj.art-47419248b4e7453badf772f28ed404bf2023-11-18T01:46:12ZengMDPI AGInternational Journal of Molecular Sciences1661-65961422-00672023-05-012410903310.3390/ijms24109033Identification of RNA-Binding Protein Targets with HyperTRIBE in <i>Saccharomyces cerevisiae</i>Weilan Piao0Chong Li1Pengkun Sun2Miaomiao Yang3Yansong Ding4Wei Song5Yunxiao Jia6Liqun Yu7Yanming Lu8Hua Jin9Key Laboratory of Molecular Medicine and Biotherapy, School of Life Science, Beijing Institute of Technology, No. 5 South Zhongguancun Street, Beijing 100081, ChinaKey Laboratory of Molecular Medicine and Biotherapy, School of Life Science, Beijing Institute of Technology, No. 5 South Zhongguancun Street, Beijing 100081, ChinaKey Laboratory of Molecular Medicine and Biotherapy, School of Life Science, Beijing Institute of Technology, No. 5 South Zhongguancun Street, Beijing 100081, ChinaKey Laboratory of Molecular Medicine and Biotherapy, School of Life Science, Beijing Institute of Technology, No. 5 South Zhongguancun Street, Beijing 100081, ChinaKey Laboratory of Molecular Medicine and Biotherapy, School of Life Science, Beijing Institute of Technology, No. 5 South Zhongguancun Street, Beijing 100081, ChinaKey Laboratory of Molecular Medicine and Biotherapy, School of Life Science, Beijing Institute of Technology, No. 5 South Zhongguancun Street, Beijing 100081, ChinaKey Laboratory of Molecular Medicine and Biotherapy, School of Life Science, Beijing Institute of Technology, No. 5 South Zhongguancun Street, Beijing 100081, ChinaKey Laboratory of Molecular Medicine and Biotherapy, School of Life Science, Beijing Institute of Technology, No. 5 South Zhongguancun Street, Beijing 100081, ChinaKey Laboratory of Molecular Medicine and Biotherapy, School of Life Science, Beijing Institute of Technology, No. 5 South Zhongguancun Street, Beijing 100081, ChinaKey Laboratory of Molecular Medicine and Biotherapy, School of Life Science, Beijing Institute of Technology, No. 5 South Zhongguancun Street, Beijing 100081, ChinaAs a master regulator in cells, RNA-binding protein (RBP) plays critical roles in organismal development, metabolism and various diseases. It regulates gene expression at various levels mostly by specific recognition of target RNA. The traditional CLIP-seq method to detect transcriptome-wide RNA targets of RBP is less efficient in yeast due to the low UV transmissivity of their cell walls. Here, we established an efficient HyperTRIBE (Targets of RNA-binding proteins Identified By Editing) in yeast, by fusing an RBP to the hyper-active catalytic domain of human RNA editing enzyme ADAR2 and expressing the fusion protein in yeast cells. The target transcripts of RBP were marked with new RNA editing events and identified by high-throughput sequencing. We successfully applied HyperTRIBE to identifying the RNA targets of two yeast RBPs, KHD1 and BFR1. The antibody-free HyperTRIBE has competitive advantages including a low background, high sensitivity and reproducibility, as well as a simple library preparation procedure, providing a reliable strategy for RBP target identification in <i>Saccharomyces cerevisiae</i>.https://www.mdpi.com/1422-0067/24/10/9033HyperTRIBERNA-binding protein (RBP)yeastKHD1BFR1 |
| spellingShingle | Weilan Piao Chong Li Pengkun Sun Miaomiao Yang Yansong Ding Wei Song Yunxiao Jia Liqun Yu Yanming Lu Hua Jin Identification of RNA-Binding Protein Targets with HyperTRIBE in <i>Saccharomyces cerevisiae</i> International Journal of Molecular Sciences HyperTRIBE RNA-binding protein (RBP) yeast KHD1 BFR1 |
| title | Identification of RNA-Binding Protein Targets with HyperTRIBE in <i>Saccharomyces cerevisiae</i> |
| title_full | Identification of RNA-Binding Protein Targets with HyperTRIBE in <i>Saccharomyces cerevisiae</i> |
| title_fullStr | Identification of RNA-Binding Protein Targets with HyperTRIBE in <i>Saccharomyces cerevisiae</i> |
| title_full_unstemmed | Identification of RNA-Binding Protein Targets with HyperTRIBE in <i>Saccharomyces cerevisiae</i> |
| title_short | Identification of RNA-Binding Protein Targets with HyperTRIBE in <i>Saccharomyces cerevisiae</i> |
| title_sort | identification of rna binding protein targets with hypertribe in i saccharomyces cerevisiae i |
| topic | HyperTRIBE RNA-binding protein (RBP) yeast KHD1 BFR1 |
| url | https://www.mdpi.com/1422-0067/24/10/9033 |
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