Mouse TU tagging: a chemical/genetic intersectional method for purifying cell type-specific nascent RNA

Miller, Michael R.; Ventura, Patrick; Devasthali, Vidusha; Vue, Zer; Thompson, Heather L.; Temple, Sally; Zong, Hui; Cleary, ML; Stankunas, Kryn; Doe, Chris Q.

doi:10.1101/gad.205278.112

Cited by 110 publications

(173 citation statements)

References 37 publications

(51 reference statements)

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“…Recently, a complementary approach to isolating RNA from cells labeled by Cre was reported, in which Cre-activated transgenic expression of uracil phosphoribosyltransferase (UPRT) permits selective RNA labeling by thiouracil (TU tagging) (7). A strength of the TU-tagging strategy is the enhanced temporal resolution for active transcription.…”

Section: Discussionmentioning

confidence: 99%

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Interrogating translational efficiency and lineage-specific transcriptomes using ribosome affinity purification

Zhou

Zhang

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

122

130

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Transcriptional profiling is a useful strategy to study development and disease. Approaches to isolate RNA from specific cell types, or from specific cellular compartments, would extend the power of this strategy. Previous work has shown that isolation of genetically tagged ribosomes (translating ribosome affinity purification; TRAP) is an effective means to isolate ribosome-bound RNA selectively from transgene-expressing cells. However, widespread application of this technology has been limited by available transgenic mouse lines. Here we characterize a TRAP allele (Rosa26 fsTRAP ) that makes this approach more widely accessible. We show that endothelium-specific activation of Rosa26 fsTRAP identifies endothelial cell-enriched transcripts, and that cardiomyocyte-restricted TRAP is a useful means to identify genes that are differentially expressed in cardiomyocytes in a disease model. Furthermore, we show that TRAP is an effective means for studying translational regulation, and that several nuclear-encoded mitochondrial genes are under strong translational control. Our analysis of ribosome-bound transcripts also shows that a subset of long intergenic noncoding RNAs are weakly ribosome-bound, but that the majority of noncoding RNAs, including most long intergenic noncoding RNAs, are ribosome-bound to the same extent as coding transcripts. Together, these data show that the TRAP strategy and the Rosa26 fsTRAP allele will be useful tools to probe cell type-specific transcriptomes, study translational regulation, and probe ribosome binding of noncoding RNAs.heart | pressure overload G enome-wide and unbiased measurement of RNA transcript levels using microarrays and RNA-seq (1) has powered fundamental advances in biology over the past decade. However, when used to study tissues composed of multiple cell types, RNA expression profiling faces two fundamental limitations. First, whole-tissue transcript levels represent the average of the distinct cell lineages in the tissue, and this averaging process can result in loss of important information or misassignment of gene expression changes in one type of cell to another. Second, RNA profiling measures transcript abundance, but translational regulation is also an important determinant of gene expression (2, 3).To overcome these limitations, approaches have been developed to isolate RNAs from selected cell types and/or selected transcript fractions (4-7). Often these approaches involve tissue dissociation followed by FACS, but this is slow and the dissociation procedure itself likely alters expression profiles. Translating ribosome affinity purification (TRAP) permits isolation of transcripts from selected cell types of intact tissues, without dissociation (4). In this approach, ribosomes of selected tissues are genetically labeled by transgenic expression of GFP fused to L10a, an integral component of the 60S ribosomal subunit. To collect RNA from the transgene-expressing subpopulation of a tissue, whole-cell lysates are prepared under conditions that stabilize ribosomes...

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Section: Discussionmentioning

confidence: 99%

“…To overcome these limitations, approaches have been developed to isolate RNAs from selected cell types and/or selected transcript fractions (4)(5)(6)(7). Often these approaches involve tissue dissociation followed by FACS, but this is slow and the dissociation procedure itself likely alters expression profiles.…”

mentioning

confidence: 99%

Interrogating translational efficiency and lineage-specific transcriptomes using ribosome affinity purification

Zhou

Zhang

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

122

130

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“…We found that DACH1 could fulfill this purpose. This homolog to the Drosophila protein Dachshund was initially identified in a screen that used Creinducible TU-tagging of RNA to discover endothelial-specific genes from whole hearts (Gay et al, 2013). Commercially available antibodies to DACH1 are specific, as evidenced by the absence of immunostaining in hearts from knockout animals (supplementary material Fig.…”

Section: Apjcreer Can Be Used To Lineage Trace Sv-derived Coronary Vementioning

confidence: 99%

The sinus venosus contributes to coronary vasculature through VEGFC-stimulated angiogenesis

et al. 2014

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Identifying coronary artery progenitors and their developmental pathways could inspire novel regenerative treatments for heart disease. Multiple sources of coronary vessels have been proposed, including the sinus venosus (SV), endocardium and proepicardium, but their relative contributions to the coronary circulation and the molecular mechanisms regulating their development are poorly understood. We created an ApjCreER mouse line as a lineagetracing tool to map SV-derived vessels onto the heart and compared the resulting lineage pattern with endocardial and proepicardial contributions to the coronary circulation. The data showed a striking compartmentalization to coronary development. ApjCreER-traced vessels contributed to a large number of arteries, capillaries and veins on the dorsal and lateral sides of the heart. By contrast, untraced vessels predominated in the midline of the ventral aspect and ventricular septum, which are vessel populations primarily derived from the endocardium. The proepicardium gave rise to a smaller fraction of vessels spaced relatively uniformly throughout the ventricular walls. Dorsal (SV-derived) and ventral (endocardialderived) coronary vessels developed in response to different growth signals. The absence of VEGFC, which is expressed in the epicardium, dramatically inhibited dorsal and lateral coronary growth but left vessels on the ventral side unaffected. We propose that complementary SV-derived and endocardial-derived migratory routes unite to form the coronary vasculature and that the former requires VEGFC, revealing its role as a tissue-specific mediator of blood endothelial development.

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“…Genetically encoded phosphoribosyltransferase (UPRT) biosynthetically labels nascent RNA in the target cell type when provided with 4-thiouracil. Chemically modified RNA molecules can then be affinity purified, a technique called TU-tagging (Miller et al, 2009;Gay et al, 2013). Alternatively, mRNA-binding proteins can be affinity tagged in specific cells and used as anchors to co-purify associated mRNAs.…”

Section: Introductionmentioning

confidence: 99%

Translational profiling through biotinylation of tagged ribosomes in zebrafish

et al. 2014

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Heterogeneity within a population of cells of the same type is a common theme in metazoan biology. Dissecting complex developmental and physiological processes crucially relies on our ability to probe the expression profile of these cell subpopulations. Current strategies rely on cell enrichment based on sequential or simultaneous use of multiple intersecting markers starting from a heterogeneous cell suspension. The extensive tissue manipulations required to generate single-cell suspensions, as well as the complexity of the required equipment, inherently complicate these approaches. Here, we propose an alternative methodology based on a genetically encoded system in the model organism Danio rerio (zebrafish). In transgenic fish, we take advantage of the combinatorial biotin transfer system, where polysome-associated mRNAs are selectively recovered from cells expressing both a tagged ribosomal subunit, Rpl10a, and the bacterial biotin ligase BirA. We have applied this technique to skeletal muscle development and identified new genes with interesting temporal expression patterns. Through this work we have thus developed additional tools for highly specific gene expression profiling.

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Mouse TU tagging: a chemical/genetic intersectional method for purifying cell type-specific nascent RNA

Cited by 110 publications

References 37 publications

Interrogating translational efficiency and lineage-specific transcriptomes using ribosome affinity purification

Interrogating translational efficiency and lineage-specific transcriptomes using ribosome affinity purification

The sinus venosus contributes to coronary vasculature through VEGFC-stimulated angiogenesis

Translational profiling through biotinylation of tagged ribosomes in zebrafish

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