EC-tagging allows cell type-specific RNA analysis
Purification of cell type-specific RNAs remains a significant challenge. One solution involves biosynthetic tagging of target RNAs. RNA tagging via incorporation of 4-thiouracil (TU) in cells expressing transgenic uracil phosphoribosyltransferase (UPRT), a method known as TU-tagging, has been used i...
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Published in | Nucleic acids research Vol. 45; no. 15; p. e138 |
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Main Authors | , , , , , , , , |
Format | Journal Article |
Language | English |
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England
Oxford University Press
06.09.2017
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Abstract | Purification of cell type-specific RNAs remains a significant challenge. One solution involves biosynthetic tagging of target RNAs. RNA tagging via incorporation of 4-thiouracil (TU) in cells expressing transgenic uracil phosphoribosyltransferase (UPRT), a method known as TU-tagging, has been used in multiple systems but can have limited specificity due to endogenous pathways of TU incorporation. Here, we describe an alternative method that requires the activity of two enzymes: cytosine deaminase (CD) and UPRT. We found that the sequential activity of these enzymes converts 5-ethynylcytosine (EC) to 5-ethynyluridine monophosphate that is subsequently incorporated into nascent RNAs. The ethynyl group allows efficient detection and purification of tagged RNAs. We show that 'EC-tagging' occurs in tissue culture cells and Drosophila engineered to express CD and UPRT. Additional control can be achieved through a split-CD approach in which functional CD is reconstituted from independently expressed fragments. We demonstrate the sensitivity and specificity of EC-tagging by obtaining cell type-specific gene expression data from intact Drosophila larvae, including transcriptome measurements from a small population of central brain neurons. EC-tagging provides several advantages over existing techniques and should be broadly useful for investigating the role of differential RNA expression in cell identity, physiology and pathology. |
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AbstractList | Purification of cell type-specific RNAs remains a significant challenge. One solution involves biosynthetic tagging of target RNAs. RNA tagging via incorporation of 4-thiouracil (TU) in cells expressing transgenic uracil phosphoribosyltransferase (UPRT), a method known as TU-tagging, has been used in multiple systems but can have limited specificity due to endogenous pathways of TU incorporation. Here, we describe an alternative method that requires the activity of two enzymes: cytosine deaminase (CD) and UPRT. We found that the sequential activity of these enzymes converts 5-ethynylcytosine (EC) to 5-ethynyluridine monophosphate that is subsequently incorporated into nascent RNAs. The ethynyl group allows efficient detection and purification of tagged RNAs. We show that 'EC-tagging' occurs in tissue culture cells and Drosophila engineered to express CD and UPRT. Additional control can be achieved through a split-CD approach in which functional CD is reconstituted from independently expressed fragments. We demonstrate the sensitivity and specificity of EC-tagging by obtaining cell type-specific gene expression data from intact Drosophila larvae, including transcriptome measurements from a small population of central brain neurons. EC-tagging provides several advantages over existing techniques and should be broadly useful for investigating the role of differential RNA expression in cell identity, physiology and pathology. Purification of cell type-specific RNAs remains a significant challenge. One solution involves biosynthetic tagging of target RNAs. RNA tagging via incorporation of 4-thiouracil (TU) in cells expressing transgenic uracil phosphoribosyltransferase (UPRT), a method known as TU-tagging, has been used in multiple systems but can have limited specificity due to endogenous pathways of TU incorporation. Here, we describe an alternative method that requires the activity of two enzymes: cytosine deaminase (CD) and UPRT. We found that the sequential activity of these enzymes converts 5-ethynylcytosine (EC) to 5-ethynyluridine monophosphate that is subsequently incorporated into nascent RNAs. The ethynyl group allows efficient detection and purification of tagged RNAs. We show that ‘EC-tagging’ occurs in tissue culture cells and Drosophila engineered to express CD and UPRT. Additional control can be achieved through a split-CD approach in which functional CD is reconstituted from independently expressed fragments. We demonstrate the sensitivity and specificity of EC-tagging by obtaining cell type-specific gene expression data from intact Drosophila larvae, including transcriptome measurements from a small population of central brain neurons. EC-tagging provides several advantages over existing techniques and should be broadly useful for investigating the role of differential RNA expression in cell identity, physiology and pathology. |
Author | Aboukilila, Mohamed Y Greenberg, Marc M Cleary, Michael D Paul, Rakesh Beasley, Samantha Burow, Dana A Hida, Naoki Fazio, Michael Spitale, Robert C |
AuthorAffiliation | 2 Department of Chemistry, Johns Hopkins University, Baltimore, MD 21218, USA 3 Department of Pharmaceutical Sciences and Department of Chemistry, University of California, Irvine, CA 92697, USA 1 Molecular and Cell Biology Unit, Quantitative and Systems Biology Graduate Program, University of California, Merced, CA 95343, USA |
AuthorAffiliation_xml | – name: 3 Department of Pharmaceutical Sciences and Department of Chemistry, University of California, Irvine, CA 92697, USA – name: 2 Department of Chemistry, Johns Hopkins University, Baltimore, MD 21218, USA – name: 1 Molecular and Cell Biology Unit, Quantitative and Systems Biology Graduate Program, University of California, Merced, CA 95343, USA |
Author_xml | – sequence: 1 givenname: Naoki surname: Hida fullname: Hida, Naoki organization: Molecular and Cell Biology Unit, Quantitative and Systems Biology Graduate Program, University of California, Merced, CA 95343, USA – sequence: 2 givenname: Mohamed Y surname: Aboukilila fullname: Aboukilila, Mohamed Y organization: Molecular and Cell Biology Unit, Quantitative and Systems Biology Graduate Program, University of California, Merced, CA 95343, USA – sequence: 3 givenname: Dana A surname: Burow fullname: Burow, Dana A organization: Molecular and Cell Biology Unit, Quantitative and Systems Biology Graduate Program, University of California, Merced, CA 95343, USA – sequence: 4 givenname: Rakesh surname: Paul fullname: Paul, Rakesh organization: Department of Chemistry, Johns Hopkins University, Baltimore, MD 21218, USA – sequence: 5 givenname: Marc M surname: Greenberg fullname: Greenberg, Marc M organization: Department of Chemistry, Johns Hopkins University, Baltimore, MD 21218, USA – sequence: 6 givenname: Michael surname: Fazio fullname: Fazio, Michael organization: Department of Pharmaceutical Sciences and Department of Chemistry, University of California, Irvine, CA 92697, USA – sequence: 7 givenname: Samantha surname: Beasley fullname: Beasley, Samantha organization: Department of Pharmaceutical Sciences and Department of Chemistry, University of California, Irvine, CA 92697, USA – sequence: 8 givenname: Robert C surname: Spitale fullname: Spitale, Robert C organization: Department of Pharmaceutical Sciences and Department of Chemistry, University of California, Irvine, CA 92697, USA – sequence: 9 givenname: Michael D surname: Cleary fullname: Cleary, Michael D organization: Molecular and Cell Biology Unit, Quantitative and Systems Biology Graduate Program, University of California, Merced, CA 95343, USA |
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SubjectTerms | Animals Animals, Genetically Modified Cell Lineage - genetics Cells, Cultured Cytosine - analogs & derivatives Cytosine - metabolism Cytosine - pharmacology Cytosine Deaminase - metabolism Drosophila melanogaster Gene Expression Profiling - methods HeLa Cells Humans Methods Online Organ Specificity - genetics Pentosyltransferases - metabolism RNA - analysis RNA - genetics Staining and Labeling - methods |
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