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Interrogating translating RNAs and cell-type specific gene expression using Cre-activated translating ribosome affinity purification

ABSTRACT: We characterize a Cre-activated TRAP allele (Rosa26fsTRAP) to show that endothelium-specific activation of Rosa26fsTRAP 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. We also show that TRAP is an effective means to study translational regulation, and we several nuclear-encoded mitochondrial genes are under strong translational control. These RNA-seq data show that the TRAP strategy and the Rosa26fsTRAP allele will be useful tools to probe cell type specific transcriptomes, and to study translational regulation.

ORGANISM(S): Mus musculus

PROVIDER: GSE45152 | GEO | 2013/08/20

SECONDARY ACCESSION(S): PRJNA193343

REPOSITORIES: GEO

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Interrogating translating RNAs and cell-type specific gene expression using Cre-activated translating ribosome affinity purification

Project description:We characterize a Cre-activated TRAP allele (Rosa26fsTRAP) to show that endothelium-specific activation of Rosa26fsTRAP 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. We also show that TRAP is an effective means to study translational regulation, and we several nuclear-encoded mitochondrial genes are under strong translational control. These RNA-seq data show that the TRAP strategy and the Rosa26fsTRAP allele will be useful tools to probe cell type specific transcriptomes, and to study translational regulation. Study TRAP RNA and total RNA in two cases: heart specific tissues, Banding or Sham, using RNA-seq technology.

2013-08-20 | E-GEOD-45152 | biostudies-arrayexpress

Translational profiling of zebrafish retina cells using translating ribosome affinity purification methods

Project description:Translating ribosome affinity purification (TRAP) methods allow cell-specific recovery of polyribosome-associated RNAs by genetic tagging of ribosomes in selected cell populations. In this study, we purified and analysed the polyribosome-associated RNA fraction of zebrafish embryo´s retinas at 22 hours post-fertilization. To this aim, we generated a transgenic strain in which the tagged ribosomes expression is restricted to the retina cells due to the activity of a specific promoter.

2021-12-29 | GSE192511 | GEO

Examining efficiency of enrichment of kidney pericyte-specific messages by TRAP (Translating Ribosome Affinity Purification)

Project description:The long term goal is to define the transcriptional changes that accompany pericyte-to-myofibroblast transition in fibrotic kidney disease. Medullary pericytes are identified by their expression of a eGFPL10a fusion protein whose expression is driven by a Col1a1 promoter. Pericyte-specific RNA is generated by eGFP-affinity purification of polysomes from medullary lysates and then subject to microarray analysis. Col1a1-eGFPL10a mice were subject to Sham or unilateral ureteral obstruction surgery. Sham kidneys were collected at day 0, and UUO kidneys were collected at day 2 or day 5 for TRAP.

2014-03-01 | E-GEOD-50439 | biostudies-arrayexpress

mRNA sequencing of samples coming from Translating Ribosome Affinity Purification (TRAP) using MSH1P::FLAG::RPL18

Project description:Col-0 transgenic plants expressing MSH1P::FLAG::RPL18 were generated in both msh1 homozygous mutant (SAIL-877-F01) and wild type backgrounds with cloned MSH1 sequence described in Xu et al. (2011) and with transformation by the floral dip method (Clough and Bent, 1998). Positive transgenic lines were screened for FLAG tag by immunoblotting methods. Polysomal mRNA from MSH1-specific cells was obtained by ribosome purification using the TRAP method essentially as reported by (Reynoso et al., 2015) from floral stem tissues.

2018-12-26 | GSE114199 | GEO

Cell-type-specific gene expression profiling using ribosome affinity purification

Project description:Post-transcriptional regulation including mRNA binding to ribosomes plays an important role in determining cell-type-specific gene expression patterns. Here, we applied an approach that profiles cell-type-specific mRNAs. The Translating Ribosome Affinity Purification method (TRAP; Heiman et al., Cell, 2008 and Doyle et al., Cell, 2008) was developed in mice and has been combined with the UAS/Gal4 system in Drosophila (Thomas et al., PLoS ONE, 2012). TRAP is a powerful method to find cell-type-specific differences at the level of the 'translatome' (Dougherty, Schmidt, Nakajima, & Heintz, Nucleic Acids Research, 2010). In parallel to published efforts, we developed and implemented the method for the fly and compared distinct head cell types and identified cell-type-specific transcript classes with neuronal (e.g. receptor-, neuropeptide- or hormone activity) or glial function (e.g. transporter activity). Neuronal TRAP genes are over-represented in the brain, larval CNS and thoracico-abdominal ganglion (Chintapalli, Wang, & Dow, Nature Genetics, 2007). Using cell-type-to-cell-type comparisons (e.g. neurons vs. glia), instead of a given cell population to the total (e.g. neurons vs. head), the differences could be identified with greater resolution. TRAP uncovered more neuronal genes compared to neuronal RNA polymerase II ChIP-seq data (Schauer et al., Cell Reports, 2013). Thus, TRAP data confirm the importance of post-transcriptional regulation in defining cell identity. TRAP is one of the best methods to reveal differential "omics" data among distinct cell types by profiling ribosome-bound mRNAs. TRAP is a promising tool to reveal cell-type-specific transcriptional and translational changes in a perturbed environment.

2017-02-01 | GSE89993 | GEO

Translating ribosome affinity purification analysis of dopaminergic neurons of the mouse ventral midbrain with age and alpha-synuclein overexpression.

Project description:To investigate the cell type-specific expression features of dopaminergic neurons in ventral midbrain, we generated DAT-TRAP mice. We then performed gene expression profiling analysis using data obtained from RNA-seq of samples collected from 3- and 18-month old mice. OVX were also bred to overexpress human alpha-synuclein. Short and long-read sequencing were performed.

2023-10-01 | GSE215276 | GEO

Cell type-specific mRNA purification by translating ribosome affinity purification (TRAP).

Project description:Cellular diversity and architectural complexity create barriers to understanding the function of the mammalian CNS at a molecular level. To address this problem, we have recently developed a methodology that provides the ability to profile the entire translated mRNA complement of any genetically defined cell population. This methodology, which we termed translating ribosome affinity purification, or TRAP, combines cell type-specific transgene expression with affinity purification of translating ribosomes. TRAP can be used to study the cell type-specific mRNA profiles of any genetically defined cell type, and it has been used in organisms ranging from Drosophila melanogaster to mice and human cultured cells. Unlike other methodologies that rely on microdissection, cell panning or cell sorting, the TRAP methodology bypasses the need for tissue fixation or single-cell suspensions (and the potential artifacts that these treatments introduce) and reports on mRNAs in the entire cell body. This protocol provides a step-by-step guide to implement the TRAP methodology, which takes 2 d to complete once all materials are in hand.

| S-EPMC4102313 | biostudies-literature

Translational profiling of zebrafish retina cells using translating ribosome affinity purification methods

Project description:Translational profiling of zebrafish retina cells using translating ribosome affinity purification methods

| PRJNA791684 | ENA

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

Project description: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.

| S-EPMC3780890 | biostudies-literature

Analysis of FGF20-regulated genes in differentiating cochlear hair and supporting cell progenitors via ribosome affinity purification

Project description:Background: Understanding the developmental mechanisms that regulate hair cell differentiation in the cochlea is essential to designing genetic therapies for acquired hearing loss due to hair cell loss or damage. We have previously identified Fibroblast Growth Factor 20 (FGF20) as having a key role in hair cell and supporting cell differentiation and patterning in the mouse cochlear sensory epithelium. To investigate the genetic landscape regulated by FGF20 signaling in hair cell and supporting cell progenitors, we employ Translating Ribosome Affinity Purification (TRAP) combined with Next Generation mRNA Sequencing (TRAPseq). Methods: In mice, we used Fgf20-Cre to activate ROSA-fsTRAP in hair cell and supporting cell progenitors, and then collected translating mRNA using TRAP from Fgf20+/- (control) and Fgf20-/- cochleae. Library preparation and sequencing were done in two separate experiments, each with 12 samples that included 2 pre-TRAP (pre-immunoprecipitation) Fgf20+/- samples, 2 TRAP Fgf20+/- samples, 4 pre-TRAP Fgf20-/- samples, and 4 TRAP Fgf20-/- samples. Samples were sequenced via Illumina HiSeq 3000. We compared pre-TRAP (pre-immunoprecipitation) samples with TRAP mRNA samples to validate the TRAP technique as well as identify genes enriched in our target cell population. We also compared Fgf20+/- and Fgf20-/- TRAP mRNA samples to identify differentially expressed genes downstream of FGF20 during hair cell and supporting cell differentiation. Results: TRAPseq targeting the prosensory cell population effectively enriches for translating mRNA within this rare cell population. TRAPseq comparing Fgf20+/- and Fgf20-/- samples identified differentially expressed genes downstream of FGF20. These included FGF-response genes Etv4, Etv5, Etv1, and Dusp6, as well as genes associated with cochlea development and hearing, such as Hey1, Hey2, Heyl, Tectb, Fat3, Cpxm2, Sall1, and cell cycle regulators such as Cdc20.

2020-04-10 | GSE148380 | GEO

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