Acrylonitrile‐Mediated Nascent RNA Sequencing for Transcriptome‐Wide Profiling of Cellular RNA Dynamics
Abstract RNA sequencing has greatly facilitated gene expression studies but is weak in studying temporal RNA dynamics; this issue can be addressed by analyzing nascent RNAs. A famous method for nascent RNA analysis is metabolic labeling with noncanonic nucleoside followed by affinity purification, h...
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| Format: | Article |
| Language: | English |
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Wiley
2020-04-01
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| Series: | Advanced Science |
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| Online Access: | https://doi.org/10.1002/advs.201900997 |
| _version_ | 1818525926955155456 |
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| author | Yuqi Chen Fan Wu Zonggui Chen Zhiyong He Qi Wei Weiwu Zeng Kun Chen Feng Xiao Yushu Yuan Xiaocheng Weng Yu Zhou Xiang Zhou |
| author_facet | Yuqi Chen Fan Wu Zonggui Chen Zhiyong He Qi Wei Weiwu Zeng Kun Chen Feng Xiao Yushu Yuan Xiaocheng Weng Yu Zhou Xiang Zhou |
| author_sort | Yuqi Chen |
| collection | DOAJ |
| description | Abstract RNA sequencing has greatly facilitated gene expression studies but is weak in studying temporal RNA dynamics; this issue can be addressed by analyzing nascent RNAs. A famous method for nascent RNA analysis is metabolic labeling with noncanonic nucleoside followed by affinity purification, however, purification processes can always introduce biases into data analysis. Here, a chemical method for nascent RNA sequencing that avoids affinity purification based on acrylonitrile‐mediated uridine‐to‐cytidine (U‐to‐C) conversion (AMUC‐seq) via 4‐thiouridine (s4U) cyanoethylation is presented. This method converts s4U base‐pairing with guanine through the nucleophilic addition of s4U to acrylonitrile. The high reaction efficiency permits AMUC‐seq directly and efficiently to recover nascent RNA information from total RNAs. AMUC‐seq is validated by being used to detect mRNA half‐lives and investigating the direct gene targets of a G‐quadruplex stabilizer, which can be regarded as potential anticancer drug, in human cells. Thousands of direct gene targets of this drug are verified (these genes are significantly enriched in cancer such as SRC and HRAS). AMUC‐seq also confirms G‐quadruplex stabilization that impacts RNA polyadenylation. These results show AMUC‐seq is qualified for the study of temporal RNA dynamics, and it can be a promising strategy to study the therapeutic mechanism of transcription‐modulating drugs. |
| first_indexed | 2024-12-11T06:15:49Z |
| format | Article |
| id | doaj.art-b777a537f61f48ebb2fe580ff91276a8 |
| institution | Directory Open Access Journal |
| issn | 2198-3844 |
| language | English |
| last_indexed | 2024-12-11T06:15:49Z |
| publishDate | 2020-04-01 |
| publisher | Wiley |
| record_format | Article |
| series | Advanced Science |
| spelling | doaj.art-b777a537f61f48ebb2fe580ff91276a82022-12-22T01:17:59ZengWileyAdvanced Science2198-38442020-04-0178n/an/a10.1002/advs.201900997Acrylonitrile‐Mediated Nascent RNA Sequencing for Transcriptome‐Wide Profiling of Cellular RNA DynamicsYuqi Chen0Fan Wu1Zonggui Chen2Zhiyong He3Qi Wei4Weiwu Zeng5Kun Chen6Feng Xiao7Yushu Yuan8Xiaocheng Weng9Yu Zhou10Xiang Zhou11College of Chemistry and Molecular Sciences Wuhan University Wuhan 430072 P. R. ChinaCollege of Chemistry and Molecular Sciences Wuhan University Wuhan 430072 P. R. ChinaThe Institute for Advanced Studies Wuhan University Wuhan 430072 P. R. ChinaCollege of Chemistry and Molecular Sciences Wuhan University Wuhan 430072 P. R. ChinaCollege of Chemistry and Molecular Sciences Wuhan University Wuhan 430072 P. R. ChinaCollege of Chemistry and Molecular Sciences Wuhan University Wuhan 430072 P. R. ChinaCollege of Chemistry and Molecular Sciences Wuhan University Wuhan 430072 P. R. ChinaCollege of Chemistry and Molecular Sciences Wuhan University Wuhan 430072 P. R. ChinaCollege of Chemistry and Molecular Sciences Wuhan University Wuhan 430072 P. R. ChinaCollege of Chemistry and Molecular Sciences Wuhan University Wuhan 430072 P. R. ChinaThe Institute for Advanced Studies Wuhan University Wuhan 430072 P. R. ChinaCollege of Chemistry and Molecular Sciences Wuhan University Wuhan 430072 P. R. ChinaAbstract RNA sequencing has greatly facilitated gene expression studies but is weak in studying temporal RNA dynamics; this issue can be addressed by analyzing nascent RNAs. A famous method for nascent RNA analysis is metabolic labeling with noncanonic nucleoside followed by affinity purification, however, purification processes can always introduce biases into data analysis. Here, a chemical method for nascent RNA sequencing that avoids affinity purification based on acrylonitrile‐mediated uridine‐to‐cytidine (U‐to‐C) conversion (AMUC‐seq) via 4‐thiouridine (s4U) cyanoethylation is presented. This method converts s4U base‐pairing with guanine through the nucleophilic addition of s4U to acrylonitrile. The high reaction efficiency permits AMUC‐seq directly and efficiently to recover nascent RNA information from total RNAs. AMUC‐seq is validated by being used to detect mRNA half‐lives and investigating the direct gene targets of a G‐quadruplex stabilizer, which can be regarded as potential anticancer drug, in human cells. Thousands of direct gene targets of this drug are verified (these genes are significantly enriched in cancer such as SRC and HRAS). AMUC‐seq also confirms G‐quadruplex stabilization that impacts RNA polyadenylation. These results show AMUC‐seq is qualified for the study of temporal RNA dynamics, and it can be a promising strategy to study the therapeutic mechanism of transcription‐modulating drugs.https://doi.org/10.1002/advs.201900997acrylonitrileG‐quadruplexnascent RNA sequencingRNA dynamics, transcriptome‐wide profilinguridine‐to‐cytidine conversion |
| spellingShingle | Yuqi Chen Fan Wu Zonggui Chen Zhiyong He Qi Wei Weiwu Zeng Kun Chen Feng Xiao Yushu Yuan Xiaocheng Weng Yu Zhou Xiang Zhou Acrylonitrile‐Mediated Nascent RNA Sequencing for Transcriptome‐Wide Profiling of Cellular RNA Dynamics Advanced Science acrylonitrile G‐quadruplex nascent RNA sequencing RNA dynamics, transcriptome‐wide profiling uridine‐to‐cytidine conversion |
| title | Acrylonitrile‐Mediated Nascent RNA Sequencing for Transcriptome‐Wide Profiling of Cellular RNA Dynamics |
| title_full | Acrylonitrile‐Mediated Nascent RNA Sequencing for Transcriptome‐Wide Profiling of Cellular RNA Dynamics |
| title_fullStr | Acrylonitrile‐Mediated Nascent RNA Sequencing for Transcriptome‐Wide Profiling of Cellular RNA Dynamics |
| title_full_unstemmed | Acrylonitrile‐Mediated Nascent RNA Sequencing for Transcriptome‐Wide Profiling of Cellular RNA Dynamics |
| title_short | Acrylonitrile‐Mediated Nascent RNA Sequencing for Transcriptome‐Wide Profiling of Cellular RNA Dynamics |
| title_sort | acrylonitrile mediated nascent rna sequencing for transcriptome wide profiling of cellular rna dynamics |
| topic | acrylonitrile G‐quadruplex nascent RNA sequencing RNA dynamics, transcriptome‐wide profiling uridine‐to‐cytidine conversion |
| url | https://doi.org/10.1002/advs.201900997 |
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