TRAP-rc, Translating Ribosome Affinity Purification from Rare Cell Populations of &lt;em&gt;Drosophila&lt;/em&gt; Embryos

Bertin, Benjamin; Renaud, Yoan; Aradhya, Rajaguru; Jagla, Krzysztof; Junion, Guillaume

doi:10.3791/52985

Cited by 14 publications

(16 citation statements)

References 34 publications

(23 reference statements)

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“…In situ transcriptomic profiling obviate these problems but lack the necessary sensitivity for disseminated cancer cells that represent less than 1% of the tissue cell population. For example, translating ribosome affinity purification and mRNA sequencing (TRAP-Seq) (Heiman et al, 2008) is not suitable to analyze cells that constitute less than 1% of the total population (Bertin et al, 2015; Obenauf et al, 2015). Direct-enzyme-based metabolic tagging of RNA with thiouracil (TU) and ethynyl cytosine (EC) in the cells of interest are limited in sensitivity and specificity due to collateral tagging and purification of tagged RNA in cells lacking the enzymes, and requires additional in vitro biotinylation steps (Cleary et al, 2005; Gay et al, 2014; Gay et al, 2013; Miller et al, 2009; Hida et al, 2017).…”

Section: Introductionmentioning

confidence: 99%

Flura-seq identifies organ-specific metabolic adaptations during early metastatic colonization

Basnet

Tian

Ganesh

et al. 2019

eLife

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Metastasis-initiating cells dynamically adapt to the distinct microenvironments of different organs, but these early adaptations are poorly understood due to the limited sensitivity of in situ transcriptomics. We developed fluorouracil-labeled RNA sequencing (Flura-seq) for in situ analysis with high sensitivity. Flura-seq utilizes cytosine deaminase (CD) to convert fluorocytosine to fluorouracil, metabolically labeling nascent RNA in rare cell populations in situ for purification and sequencing. Flura-seq revealed hundreds of unique, dynamic organ-specific gene signatures depending on the microenvironment in mouse xenograft breast cancer micrometastases. Specifically, the mitochondrial electron transport Complex I, oxidative stress and counteracting antioxidant programs were induced in pulmonary micrometastases, compared to mammary tumors or brain micrometastases. We confirmed lung metastasis-specific increase in oxidative stress and upregulation of antioxidants in clinical samples, thus validating Flura-seq’s utility in identifying clinically actionable microenvironmental adaptations in early metastasis. The sensitivity, robustness and economy of Flura-seq are broadly applicable beyond cancer research.

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Section: Introductionmentioning

confidence: 99%

Flura-seq identifies organ-specific metabolic adaptations during early metastatic colonization

Basnet

Tian

Ganesh

et al. 2019

eLife

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“…In situ transcriptomic profiling obviate these problems but lack the necessary sensitivity for cell populations that represent less than 1% of the tissue. For example, translating ribosome affinity purification and mRNA sequencing (TRAP-Seq) (Heiman et al, 2008) is not suitable to analyze cells that constitute less than 1% of the total population (Bertin et al, 2015;Obenauf et al, 2015). Direct enzyme based metabolic tagging of RNA with thiouracil (TU) and ethynyl cytosine (EC) in the cells of interest are limited in sensitivity and specificity due to collateral tagging of RNA in cells lacking the enzymes, and requires additional in vitro biotinylation steps (Cleary et al, 2005;Miller et al, 2009;Gay et al, 2013 and2014;Hida et al, 2017).…”

mentioning

confidence: 99%

Labeling and Isolation of Fluorouracil Tagged RNA by Cytosine Deaminase Expression

Basnet¹,

Massagué²

2019

BIO-PROTOCOL

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Tissues are comprised of different cell types whose interactions elicit distinct gene expression patterns that regulate tissue formation, regeneration, homeostasis and repair. Analysis of these gene expression patterns require methods that can capture as closely as possible the transcriptomes of cells of interest in their tissue microenvironment. Current technologies designed to study in situ transcriptomics are limited by their low sensitivity that require cell types to represent more than 1% of the total tissue, making it challenging to transcriptionally profile rare cell populations rapidly isolated from their native microenvironment. To address this problem, we developed fluorouracil-tagged RNA sequencing (Flura-seq) that utilizes cytosine deaminase (CD) to convert the non-natural pyrimidine fluorocytosine to fluorouracil. Expression of S. cerevisiae CD and exposure to fluorocytosine generates fluorouracil and metabolically labels newly synthesized RNAs specifically in cells of interest. Fluorouracil-tagged RNAs can then be immunopurified and used for downstream analysis. Here, we describe the detailed protocol to perform Flura-seq both in vitro and in vivo. The robustness, simplicity and lack of toxicity of Flura-seq make this tool broadly applicable to many studies in developmental, regenerative, and cancer biology.

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“…TRAP was first developed to isolate polysome-associated mRNA from a subset of neurons in mice (Heiman et al, 2008) and was later adapted to other model organisms including Xenopus (Watson et al, 2012), zebrafish (Tryon et al, 2013) and Drosophila (Bertin et al, 2015; Thomas et al, 2012). Here we applied TRAP to determine the first translatomic signatures underlying diversification of muscle types, and we identified Gel and dCryAB as new LT muscle identity realisator genes.…”

Section: Discussionmentioning

confidence: 99%

“…Images were processed with ImageJ. TRAP experiment RPL10aGFP--tagged embryos were collected, and messenger RNAs from the different muscle populations isolated as described (18). Live imaging Lms--Gal4; UAS--lifeAct double transgenic line has been generated and used for time lapse imaging of LT muscle formation in gel mutant context.…”

Section: Spatial Heatmap From Lists Generated By the Comparison Of Smentioning

confidence: 99%

“…To further analyse diversification processes and identify genes acting downstream of iTFs, we optimised translating ribosome affinity purification (TRAP) [16] to small subsets of FCs and developing muscle precursors [17]. TRAP-purified mRNA profiling followed by bioinformatic analysis and generation of temporal transition profiles identified muscle subsetspecific translatome signatures with Gelsolin ( Gel ) and dCryAB as new identity realisator genes controlling shape- and size-related properties of Lms-expressing muscles.…”

Section: Introductionmentioning

confidence: 99%

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GelsolinanddCryABact downstream of muscle identity genes and contribute to preventing muscle splitting and branching inDrosophila

Bertin

Renaud

Jagla

et al. 2019

Preprint

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How the diversification of cell types is regulated during development to generate stereotyped tissue patterns remains a challenging question. Here we applied cell type specific translational profiling (TRAP) approach to identify gene expression signatures underlying the diversification of two muscle subsets in Drosophila. Targeting the Slou-positive muscle population, we identified Ptx1 as a new component of the ventral muscle identity code. We also generated temporal transition profiles of TRAP--enriched genes and found that a set of genes encoding regulators of the actin cytoskeleton fitted a cluster specific for lateral transverse (LT) muscles. Two of them, dCryAB and Gelsolin (Gel), encoding actin--binding sHsp and actin--severing protein respectively, displayed LT muscle prevailing expression positively regulated by LT identity factors Lms and Apterous. In dCryAB--mutant embryos, LT muscle shapes had an irregular appearance, whereas Gel--devoid LTs occasionally developed muscle fibre splitting. Importantly, split LT muscles in Gel mutants were enlarged and bore a greater number of myonuclei, a phenotype phenocopied by overexpression of the duf fusion gene. Muscle fibre splitting is a hallmark of diseased muscle in Duchenne and other muscular dystrophies, but how muscles get split is unknown. Our analyses yield evidence that an excessive myoblast fusion could result in splitting, and suggest a deregulation of fusion machinery in dystrophic muscles. We identify new muscle identity gene Ptx1 and a new class of identity realisator genes including Gel, whose function links muscle identity in Drosophila to dystrophic muscle splitting phenotype in humans. Significance statementKey features that make two cells differ from each other are shape and size. Here, by applying translational profiling of muscle subsets in Drosophila, we identify new muscle identity realisator genes dCryAB and Gel encoding actin--binding sHsp and actin--severing protein, respectively. They both regulate growth--related properties of a subset of embryonic muscles, the LT muscles. Intriguingly, Gel--devoid LTs undergo muscle fiber splitting, a phenotype also observed in Duchenne muscular dystrophy patients. LT muscle splitting in Gel mutants is due to an excessive myoblast fusion resulting in increased muscle size, offering clues to still unknown mechanisms of pathological muscle splitting in humans.

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TRAP-rc, Translating Ribosome Affinity Purification from Rare Cell Populations of <em>Drosophila</em> Embryos

Cited by 14 publications

References 34 publications

Flura-seq identifies organ-specific metabolic adaptations during early metastatic colonization

Flura-seq identifies organ-specific metabolic adaptations during early metastatic colonization

Labeling and Isolation of Fluorouracil Tagged RNA by Cytosine Deaminase Expression

GelsolinanddCryABact downstream of muscle identity genes and contribute to preventing muscle splitting and branching inDrosophila

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