Metabolic RNA Labeling and Translating Ribosome Affinity Purification for Measurement of Nascent RNA Translation
Regulation of gene expression in response to various biological processes, including extracellular stimulation and environmental adaptation, requires nascent mRNA synthesis and translation. Simultaneous analysis of the coordinated regulation of dynamic mRNA synthesis and translation using the same e...
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Bio-protocol LLC
2024-10-01
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| Online Access: | https://bio-protocol.org/en/bpdetail?id=5091&type=0 |
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| author | Hirotatsu Imai Akio Yamashita |
| author_facet | Hirotatsu Imai Akio Yamashita |
| author_sort | Hirotatsu Imai |
| collection | DOAJ |
| description | Regulation of gene expression in response to various biological processes, including extracellular stimulation and environmental adaptation, requires nascent mRNA synthesis and translation. Simultaneous analysis of the coordinated regulation of dynamic mRNA synthesis and translation using the same experiment remains a major challenge in the field. Here, we describe a step-by-step protocol for the simultaneous measurement of transcription of nascent mRNA and its translation at the gene level during the acute unfolded protein response (UPR) in HEK293 cells by combining 4-thiouridine metabolic mRNA labeling with translational ribosome affinity purification (TRAP) using a monoclonal antibody against evolutionarily conserved ribosomal P-stalk proteins (P-TRAP). Since P-TRAP captures full-length RNAs bound to ribosomes, it is compatible with 3' mRNA-seq, which analyzes the uridine-rich 3' UTRs of polyadenylated RNAs, allowing robust quantification of T>C conversions. Our nascent P-TRAP (nP-TRAP) method, in which P-TRAP is combined with metabolic mRNA labeling, can serve as a simple and powerful tool to analyze the coordinated regulation of transcription and translation of individual genes in cultured cells. |
| format | Article |
| id | doaj-art-0455e752afee4d3ba9d48c672180baa6 |
| institution | OA Journals |
| issn | 2331-8325 |
| language | English |
| publishDate | 2024-10-01 |
| publisher | Bio-protocol LLC |
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| series | Bio-Protocol |
| spelling | doaj-art-0455e752afee4d3ba9d48c672180baa62025-08-20T02:35:30ZengBio-protocol LLCBio-Protocol2331-83252024-10-01142010.21769/BioProtoc.5091Metabolic RNA Labeling and Translating Ribosome Affinity Purification for Measurement of Nascent RNA TranslationHirotatsu Imai0Akio Yamashita1Department of Investigative Medicine, University of the Ryukyus, Uehara 207, Okinawa, JapanDepartment of Investigative Medicine, University of the Ryukyus, Uehara 207, Okinawa, JapanRegulation of gene expression in response to various biological processes, including extracellular stimulation and environmental adaptation, requires nascent mRNA synthesis and translation. Simultaneous analysis of the coordinated regulation of dynamic mRNA synthesis and translation using the same experiment remains a major challenge in the field. Here, we describe a step-by-step protocol for the simultaneous measurement of transcription of nascent mRNA and its translation at the gene level during the acute unfolded protein response (UPR) in HEK293 cells by combining 4-thiouridine metabolic mRNA labeling with translational ribosome affinity purification (TRAP) using a monoclonal antibody against evolutionarily conserved ribosomal P-stalk proteins (P-TRAP). Since P-TRAP captures full-length RNAs bound to ribosomes, it is compatible with 3' mRNA-seq, which analyzes the uridine-rich 3' UTRs of polyadenylated RNAs, allowing robust quantification of T>C conversions. Our nascent P-TRAP (nP-TRAP) method, in which P-TRAP is combined with metabolic mRNA labeling, can serve as a simple and powerful tool to analyze the coordinated regulation of transcription and translation of individual genes in cultured cells.https://bio-protocol.org/en/bpdetail?id=5091&type=0 |
| spellingShingle | Hirotatsu Imai Akio Yamashita Metabolic RNA Labeling and Translating Ribosome Affinity Purification for Measurement of Nascent RNA Translation Bio-Protocol |
| title | Metabolic RNA Labeling and Translating Ribosome Affinity Purification for Measurement of Nascent RNA Translation |
| title_full | Metabolic RNA Labeling and Translating Ribosome Affinity Purification for Measurement of Nascent RNA Translation |
| title_fullStr | Metabolic RNA Labeling and Translating Ribosome Affinity Purification for Measurement of Nascent RNA Translation |
| title_full_unstemmed | Metabolic RNA Labeling and Translating Ribosome Affinity Purification for Measurement of Nascent RNA Translation |
| title_short | Metabolic RNA Labeling and Translating Ribosome Affinity Purification for Measurement of Nascent RNA Translation |
| title_sort | metabolic rna labeling and translating ribosome affinity purification for measurement of nascent rna translation |
| url | https://bio-protocol.org/en/bpdetail?id=5091&type=0 |
| work_keys_str_mv | AT hirotatsuimai metabolicrnalabelingandtranslatingribosomeaffinitypurificationformeasurementofnascentrnatranslation AT akioyamashita metabolicrnalabelingandtranslatingribosomeaffinitypurificationformeasurementofnascentrnatranslation |