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

Abstract

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.

Early dynamics of photosynthetic Lhcf2 and Lhcf15 transcription and mRNA stabilities in response to herbivory-related decadienal in Phaeodactylum tricornutum

“…The observed changes in the Lhcf2 and Lhcf15 mRNA levels in response to DD could be regulated either at the level of transcription, mRNA stability or both. In vivo metabolic labeling of RNA with 4-thiouracil (4-TU) or 4-thiouridine is an efficient method to isolate labeled newly synthesized RNA fractions for determining mRNA synthesis and decay and has been developed in a range of eukaryotes 41,42 . We used 4-TU to label newly synthesized RNA because P. tricornutum contains the gene for uracil phosphoribosyltransferase (Uniprot ID B7FUW5), an enzyme for recycling uracil to uridine monophosphate in the pyrimidine salvage pathway 43 .…”

Identification of novel regulators of dendrite arborization using cell type-specific RNA metabolic labeling

“…For example, in the embryonic nervous system, mRNAs encoding the three RBPs we selected for analysis (ythdc1, heph, and hrg) all have below average half-lives [13]. As previously described [7], metabolic RNA tagging approaches are most effective when comparing purified target cell RNA to a reference generated by metabolic tagging in all cell types of the starting material (tissue or whole animal). For this reason, any genes transcribed in more abundant cells of the carcass are unlikely to be identified as md neuronenriched in this study.…”

“…One caveat of physical isolation is that neurons are removed from their natural environment and undergo processing prior to RNA extraction, possibly inducing transcriptional and post-transcriptional responses that do not reflect in vivo gene expression. An alternative approach is to use biosynthetic RNA tagging methods that do not require physical isolation and enrich for nascent and recentlytranscribed mRNAs [7]. These methods use metabolic labeling, under in vivo conditions, to generate tagged RNAs in the cells of interest.…”

Identification of novel regulators of dendrite arborization using cell type-specific RNA metabolic labeling

“…For example, in the embryonic nervous system, mRNAs encoding the three RBPs we selected for analysis (ythdc1, heph, and hrg) all have below average half-lives [13]. As previously described [7], metabolic RNA tagging approaches are most effective when comparing purified target cell RNA to a reference generated by metabolic tagging in all cell types of the starting material (tissue or whole animal). For this reason, any genes transcribed in more abundant cells of the carcass are unlikely to be identified as md neuron-enriched in this study.…”

“…One caveat of physical isolation is that neurons are removed from their natural environment and undergo processing prior to RNA extraction, possibly inducing transcriptional and post-transcriptional responses that do not reflect in vivo gene expression. An alternative approach is to use biosynthetic RNA tagging methods that do not require physical isolation and enrich for nascent and recently-transcribed mRNAs [7]. These methods use metabolic labeling, under in vivo conditions, to generate tagged RNAs in the cells of interest.…”