Advanced RPL19-TRAPKI-seq method reveals mechanism of action of bioactive compounds

doi:10.1007/s13659-025-00500-3

Natural Products and Bioprospecting ›› 2025, Vol. 15 ›› Issue (2): 16-16.DOI: 10.1007/s13659-025-00500-3

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Advanced RPL19-TRAP^KI-seq method reveals mechanism of action of bioactive compounds

Di Zhu¹, Junchi Hu², Renke Tan³, Xiaofeng Lin³, Ruina Wang³, Junyan Lu⁴, Biao Yu⁵, Yongmei Xie⁶, Xiaohua Ni⁷, Chunmin Liang¹, Yongjun Dang², Wei Jiang³

1. Laboratory of Tumor Immunology, Department of Human Anatomy, Histology and Embryology, School of Basic Medical Sciences, Shanghai Medical College, Fudan University, Shanghai, 200032, China;
2. Basic Medicine Research and Innovation Center for Novel Target and Therapeutic Intervention, Ministry of Education, College of Pharmacy, Chongqing Medical University, Chongqing Medical University, Chongqing, 400016, China;
3. Key Laboratory of Metabolism and Molecular Medicine, Ministry of Education, Department of Biochemistry and Molecular Biology, School of Basic Medical Sciences, Shanghai Medical College, Fudan University, Shanghai, 200032, China;
4. Medical Faculty Heidelberg, Heidelberg University, Heidelberg, Germany;
5. State Key Laboratory of Bioorganic and Natural Products Chemistry, Center for Excellence in Molecular Synthesis, Shanghai Institute of Organic Chemistry, Chinese Academy of Sciences, and University of Chinese Academy of Sciences, Shanghai, 200032, China;
6. State Key Laboratory of Biotherapy and Cancer Center, West China Hospital, Sichuan University and Collaborative Innovation Center of Biotherapy, Chengdu, 610041, China;
7. Shanghai-MOST Key Laboratory of Health and Disease Genomics, NHC Key Lab of Reproduction Regulation, Shanghai Institute for Biomedical and Pharmaceutical Technologies, Shanghai, 200237, China

Received:2025-01-07 Accepted:2025-02-18 Online:2025-05-17 Published:2025-04-25
Contact: Xiaohua Ni, E-mail:xhni_sippr@163.com;Chunmin Liang, E-mail:cmliang@fudan.edu.cn;Yongjun Dang, E-mail:yjdang@cqmu.edu.cn;Wei Jiang, E-mail:jiangw@fudan.edu.cn
Supported by:
This work was supported by the National Key Research and Development Program of China (No. 2022YFC2804800 to W.J.), the National Natural Science Foundation of China (No. 22494704., 22137002 to Y.D., 92253305 to W.J. and 31971111 to C.L.), the Science and Technology Commission of Shanghai Municipality (Grant 20JC1410900 to Y.D.), the University Innovation Research Group in Chongqing (No. CXQT21016 to Y.D.), the Chongqing Talent Program Project (No. CQYC20200302119 to Y.D.), High-Level Innovation Platform Cultivation Plan of Chongqing (to Y.D.), Joint Fund of the Natural Science Innovation and Development Foundation of Chongqing (to Y.D.), Program for Professor of Special Appointment (Eastern Scholar) at Shanghai Institutions of Higher Learning (to W.J.) and Chongqing Doctoral Express Entry Project (No. CSTB2022BSXM-JCX0044 to J.H.).

Advanced RPL19-TRAP^KI-seq method reveals mechanism of action of bioactive compounds

Di Zhu¹, Junchi Hu², Renke Tan³, Xiaofeng Lin³, Ruina Wang³, Junyan Lu⁴, Biao Yu⁵, Yongmei Xie⁶, Xiaohua Ni⁷, Chunmin Liang¹, Yongjun Dang², Wei Jiang³

1. Laboratory of Tumor Immunology, Department of Human Anatomy, Histology and Embryology, School of Basic Medical Sciences, Shanghai Medical College, Fudan University, Shanghai, 200032, China;
2. Basic Medicine Research and Innovation Center for Novel Target and Therapeutic Intervention, Ministry of Education, College of Pharmacy, Chongqing Medical University, Chongqing Medical University, Chongqing, 400016, China;
3. Key Laboratory of Metabolism and Molecular Medicine, Ministry of Education, Department of Biochemistry and Molecular Biology, School of Basic Medical Sciences, Shanghai Medical College, Fudan University, Shanghai, 200032, China;
4. Medical Faculty Heidelberg, Heidelberg University, Heidelberg, Germany;
5. State Key Laboratory of Bioorganic and Natural Products Chemistry, Center for Excellence in Molecular Synthesis, Shanghai Institute of Organic Chemistry, Chinese Academy of Sciences, and University of Chinese Academy of Sciences, Shanghai, 200032, China;
6. State Key Laboratory of Biotherapy and Cancer Center, West China Hospital, Sichuan University and Collaborative Innovation Center of Biotherapy, Chengdu, 610041, China;
7. Shanghai-MOST Key Laboratory of Health and Disease Genomics, NHC Key Lab of Reproduction Regulation, Shanghai Institute for Biomedical and Pharmaceutical Technologies, Shanghai, 200237, China

通讯作者: Xiaohua Ni, E-mail:xhni_sippr@163.com;Chunmin Liang, E-mail:cmliang@fudan.edu.cn;Yongjun Dang, E-mail:yjdang@cqmu.edu.cn;Wei Jiang, E-mail:jiangw@fudan.edu.cn
基金资助:
This work was supported by the National Key Research and Development Program of China (No. 2022YFC2804800 to W.J.), the National Natural Science Foundation of China (No. 22494704., 22137002 to Y.D., 92253305 to W.J. and 31971111 to C.L.), the Science and Technology Commission of Shanghai Municipality (Grant 20JC1410900 to Y.D.), the University Innovation Research Group in Chongqing (No. CXQT21016 to Y.D.), the Chongqing Talent Program Project (No. CQYC20200302119 to Y.D.), High-Level Innovation Platform Cultivation Plan of Chongqing (to Y.D.), Joint Fund of the Natural Science Innovation and Development Foundation of Chongqing (to Y.D.), Program for Professor of Special Appointment (Eastern Scholar) at Shanghai Institutions of Higher Learning (to W.J.) and Chongqing Doctoral Express Entry Project (No. CSTB2022BSXM-JCX0044 to J.H.).

Abstract

Abstract: Natural products play a crucial role in new drug development, but their druggability is often limited by uncertain molecular targets and insufficient research on mechanisms of action. In this study, we developed a new RPL19-TRAP^KI-seq method, combining CRISPR/Cas9 and TRAP technologies, to investigate these mechanisms. We identified and validated seven ribosomal large subunit surface proteins suitable for TRAP, selecting RPL19 for its high enrichment. We successfully established a stable cell line expressing EGFP-RPL19 using CRISPR knock-in and verified its efficiency and specificity in enriching ribosomes and translating mRNA. Integrated with next-generation sequencing, this method allows precise detection of translating mRNA. We validated RPL19-TRAP^KI-seq by investigating rapamycin, an mTOR inhibitor, yielding results consistent with previous reports. This optimized TRAP technology provides an accurate representation of translating mRNA, closely reflecting protein expression levels. Furthermore, we investigated SBF-1, a 23-oxa-analog of natural saponin OSW-1 with significant anti-tumor activity but an unclear mechanism. Using RPL19-TRAP^KI-seq, we found that SBF-1 exerts its cytotoxic effects on tumor cells by disturbing cellular oxidative phosphorylation. In conclusion, our method has been proven to be a promising tool that can reveal the mechanisms of small molecules with greater accuracy, setting the stage for future exploration of small molecules and advancing the fields of pharmacology and therapeutic development.

Key words: TRAP, Ribosome profiling, SBF-1, Oxidative phosphorylation

摘要： Natural products play a crucial role in new drug development, but their druggability is often limited by uncertain molecular targets and insufficient research on mechanisms of action. In this study, we developed a new RPL19-TRAP^KI-seq method, combining CRISPR/Cas9 and TRAP technologies, to investigate these mechanisms. We identified and validated seven ribosomal large subunit surface proteins suitable for TRAP, selecting RPL19 for its high enrichment. We successfully established a stable cell line expressing EGFP-RPL19 using CRISPR knock-in and verified its efficiency and specificity in enriching ribosomes and translating mRNA. Integrated with next-generation sequencing, this method allows precise detection of translating mRNA. We validated RPL19-TRAP^KI-seq by investigating rapamycin, an mTOR inhibitor, yielding results consistent with previous reports. This optimized TRAP technology provides an accurate representation of translating mRNA, closely reflecting protein expression levels. Furthermore, we investigated SBF-1, a 23-oxa-analog of natural saponin OSW-1 with significant anti-tumor activity but an unclear mechanism. Using RPL19-TRAP^KI-seq, we found that SBF-1 exerts its cytotoxic effects on tumor cells by disturbing cellular oxidative phosphorylation. In conclusion, our method has been proven to be a promising tool that can reveal the mechanisms of small molecules with greater accuracy, setting the stage for future exploration of small molecules and advancing the fields of pharmacology and therapeutic development.

关键词: TRAP, Ribosome profiling, SBF-1, Oxidative phosphorylation

Di Zhu, Junchi Hu, Renke Tan, Xiaofeng Lin, Ruina Wang, Junyan Lu, Biao Yu, Yongmei Xie, Xiaohua Ni, Chunmin Liang, Yongjun Dang, Wei Jiang. Advanced RPL19-TRAP^KI-seq method reveals mechanism of action of bioactive compounds[J]. Natural Products and Bioprospecting, 2025, 15(2): 16-16.

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Advanced RPL19-TRAP^KI-seq method reveals mechanism of action of bioactive compounds

Advanced RPL19-TRAP^KI-seq method reveals mechanism of action of bioactive compounds

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