This Title All WIREs
How to cite this WIREs title:
WIREs Dev Biol
Impact Factor: 5.814

Uncovering cell type‐specific complexities of gene expression and RNA metabolism by TU‐tagging and EC‐tagging

Full article on Wiley Online Library:   HTML PDF

Can't access this content? Tell your librarian.

Cell type‐specific transcription is a key determinant of cell fate and function. An ongoing challenge in biology is to develop robust and stringent biochemical methods to explore gene expression with cell type specificity. This challenge has become even greater as researchers attempt to apply high‐throughput RNA analysis methods under in vivo conditions. TU‐tagging and EC‐tagging are in vivo biosynthetic RNA tagging techniques that allow spatial and temporal specificity in RNA purification. Spatial specificity is achieved through targeted expression of pyrimidine salvage enzymes (uracil phosphoribosyltransferase and cytosine deaminase) and temporal specificity is achieved by controlling exposure to bioorthogonal substrates of these enzymes (4‐thiouracil and 5‐ethynylcytosine). Tagged RNAs can be purified from total RNA extracted from an animal or tissue and used in transcriptome profiling analyses. In addition to identifying cell type‐specific mRNA profiles, these techniques are applicable to noncoding RNAs and can be used to measure RNA transcription and decay. Potential applications of TU‐tagging and EC‐tagging also include fluorescent RNA imaging and selective definition of RNA–protein interactions. TU‐tagging and EC‐tagging hold great promise for supporting research at the intersection of RNA biology and developmental biology. This article is categorized under: Technologies > Analysis of the Transcriptome
Biosynthetic RNA tagging pathways and basic RNA purification scheme. (a) Conversion of 4‐thiouracil to 4‐thiouridine monophosphate by uracil phosphoribosyltransferase (UPRT). The dashed arrow represents the phosphorylation of 4‐thiouridine monophosphate by nucleotide kinases. (b) Conversion of 5‐ethynylcytosine to 5‐ethynyluracil by cytosine deaminase (CD) and conversion of 5‐ethynyluracil to 5‐ethynyluridine monophosphate by UPRT. The dashed arrow represents the phosphorylation of 5‐ethynyluridine monophosphate by nucleotide kinases. (c) The bioorthogonal nucleotide produced in target cells (orange circle) is incorporated into nascent RNAs. Tagged RNAs are purified from a mixture of all RNAs by selective biotinylation and capture on streptavidin beads
[ Normal View | Magnified View ]
Applications beyond standard transcriptome profiling. (a) Relative transcription activity can be measured using 4‐thiouracil (4TU) or 5‐ethynylcytosine (5EC) pulse tagging. In this example, a single gene A transcript is made during the pulse compared to four gene B transcripts, indicating a higher rate of transcription at gene B. (b) RNA decay can be measured by following a 4TU or 5EC pulse with a uridine chase. Excess uridine ensures no new tagged RNAs are made during the chase and tagged RNAs are made during the pulse can be quantified during a chase timecourse. In this example, gene A mRNA is more stable (all gene B mRNAs are degraded after 3 hr). (c) Cell type‐specific RNA metabolism can be analyzed by visualizing ethynyluridine‐tagged RNAs. Fluorophore‐coupling by “click chemistry” in fixed tissue samples can be restricted to specific cell types (the CD:UPRT‐positive target cells). In this example, the middle CD:UPRT‐positive cell shows a general decrease in RNA synthesis compared to other target cells, including decreased ribosomal RNA synthesis revealed by weak nucleolar fluorescence (green circles in the nucleus). (d) Cell type‐specific RNA‐binding protein (RBP) targets can be identified by TU‐tagging combined with PAR‐CLIP. UV light enhances crosslinking of the RBP to 4‐thiouridines in target cells. Following immunoprecipitation and sequencing of RBP‐associated RNA fragments, only the sequences derived from target cells will have the characteristic thymidine to cytosine transition
[ Normal View | Magnified View ]
Reference RNA type affects TU‐tagging and EC‐tagging transcriptome profiling results. (a) Key: the target cell expressing UPRT or CD:UPRT is white, nontarget cells are green. Biosynthetically tagged mRNA is blue, nontagged mRNA is black. Distinct mRNAs (X and Y) are represented by different shapes. (b) When gene X is only transcribed in the target cell, a type I reference (containing tagged and nontagged mRNAs) is sufficient to reveal that mRNA X is target cell‐specific. (c) When gene X is transcribed in both cell types, a type I reference may fail to reveal elevated expression of gene X in the target cell. In contrast, use of a type II reference containing only newly made mRNAs reveals that gene X is transcribed at higher levels in the target cell
[ Normal View | Magnified View ]

Browse by Topic

Technologies > Analysis of the Transcriptome