ArticleNucleic acids research2017
EC-tagging allows cell type-specific RNA analysis.
Article in Nucleic acids research, 2017. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 19 papers.
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Who cites it
19 citing papers in PubMed, 43 citations in OpenAlex.
- Labeling of nascent RNA in the C. elegans intestine.PloS one · 2026Article
- A selective and sensitive detection system for 4-thiouridine modification in RNA.RNA (New York, N.Y.) · 2023Article
- mRNAs encoding neurodevelopmental regulators have equal N6-methyladenosine stoichiometry in Drosophila neuroblasts and neurons.Neural development · 2022Article
- Exploiting Endogenous Enzymes for Cancer-Cell Selective Metabolic Labeling of RNA in Vivo.Journal of the American Chemical Society · 2022Article
- I-KCKT allows dissection-free RNA profiling of adultDevelopment (Cambridge, England) · 2021Article
- A Bump-Hole Strategy for Increased Stringency of Cell-Specific Metabolic Labeling of RNA.ACS chemical biology · 2020Article
- Covalent labeling of nucleic acids.Chemical Society reviews · 2020Review
- An optimized chemical-genetic method for cell-specific metabolic labeling of RNA.Nature methods · 2020Article
- Identification of novel regulators of dendrite arborization using cell type-specific RNA metabolic labeling.PloS one · 2020Article
- Labeling and Isolation of Fluorouracil Tagged RNA by Cytosine Deaminase Expression.Bio-protocol · 2019Article
- Studying alcohol use disorder using Drosophila melanogaster in the era of 'Big Data'.Behavioral and brain functions : BBF · 2019Review
- Flura-seq identifies organ-specific metabolic adaptations during early metastatic colonization.eLife · 2019Article
- Global analysis of RNA metabolism using bio-orthogonal labeling coupled with next-generation RNA sequencing.Methods (San Diego, Calif.) · 2019Review
- Nuclear Transcriptomes of the Seven Neuronal Cell Types That Constitute theG3 (Bethesda, Md.) · 2019Article
- Protected pyrimidine nucleosides for cell-specific metabolic labeling of RNA.Tetrahedron letters · 2018Article
- Drosophila nucleostemin 3 is required to maintain larval neuroblast proliferation.Developmental biology · 2018Article
- Bioorthogonal Metabolic Labeling of Nascent RNA in Neurons Improves the Sensitivity of Transcriptome-Wide Profiling.ACS chemical neuroscience · 2018Article
- SLAM-ITseq: sequencing cell type-specific transcriptomes without cell sorting.Development (Cambridge, England) · 2018Article
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Authors and funding
9 authors at 3 institutions in 1 country.
Funding
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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