Cell type-specific transcriptome profiling in C. elegans using the Translating Ribosome Affinity Purification technique

Gracida, Xicotencatl; Calarco, John A.

doi:10.1016/j.ymeth.2017.06.023

Cited by 26 publications

(23 citation statements)

References 33 publications

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“…Previously, global tissue-specific isoform expression has been difficult to assess because most experiments have used whole worms, have detected gene expression using isoform-indiscriminant microarray platforms, require affinity purification [ 38 ], or only utilize short reads biased toward the 3’ ends of mRNA [ 10 ]. Here we used relatively long reads with good coverage through most gene bodies ( S1B Fig ), and thus were able to assess the levels of expression of individual exons in each tissue.…”

Section: Resultsmentioning

confidence: 99%

Transcriptome analysis of adult Caenorhabditis elegans cells reveals tissue-specific gene and isoform expression

et al. 2018

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The biology and behavior of adults differ substantially from those of developing animals, and cell-specific information is critical for deciphering the biology of multicellular animals. Thus, adult tissue-specific transcriptomic data are critical for understanding molecular mechanisms that control their phenotypes. We used adult cell-specific isolation to identify the transcriptomes of C. elegans’ four major tissues (or “tissue-ome”), identifying ubiquitously expressed and tissue-specific “enriched” genes. These data newly reveal the hypodermis’ metabolic character, suggest potential worm-human tissue orthologies, and identify tissue-specific changes in the Insulin/IGF-1 signaling pathway. Tissue-specific alternative splicing analysis identified a large set of collagen isoforms. Finally, we developed a machine learning-based prediction tool for 76 sub-tissue cell types, which we used to predict cellular expression differences in IIS/FOXO signaling, stage-specific TGF-β activity, and basal vs. memory-induced CREB transcription. Together, these data provide a rich resource for understanding the biology governing multicellular adult animals.

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Section: Resultsmentioning

confidence: 99%

Transcriptome analysis of adult Caenorhabditis elegans cells reveals tissue-specific gene and isoform expression

et al. 2018

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“…One of the latest versions of this method is referred to as polyA-tagging and sequencing (PAT-seq; Blazie et al 2015Blazie et al , 2017.The PAB-based method is technically demanding, in particular for the analysis of a small number of cells (Takayama et al 2010) and has been shown to be associated with significant background noise (Von Stetina et al 2007;Ma et al 2016). Two more recent alternatives to PAB-based mRNA tagging are tissue-specific Translating Ribosome Affinity Purification (TRAP; Gracida and Calarco 2017;Rhoades et al 2019) and trans-splicing-based RNA tagging (SRT; Ma et al 2016). TRAP analysis focuses on ribosome-engaged mRNAs recovered after cross-linking and immunoprecipitation of a tagged ribosome subunit.…”

Section: Introductionmentioning

confidence: 99%

Tissue-Specific Transcription Footprinting Using RNA PoI DamID (RAPID) in Caenorhabditis elegans

et al. 2020

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Differential gene expression across cell types underlies the development and cell physiology in multicellular organisms. C. elegans is a powerful, extensively used model to address these biological questions. A remaining bottleneck relates, however, to the difficulty to obtain comprehensive tissue-specific gene transcription data, since available methods are still challenging to execute and/or require large worm populations. Here, we introduce the RNAPol DamID (RAPID) approach, in which the Dam methyltransferase is fused to a ubiquitous RNA polymerase subunit in order to create transcriptional footprints via methyl marks on the DNA of transcribed genes. To validate the method, we determined the polymerase footprints in whole animals, sorted embryonic blastomeres and in different tissues from intact young adults by driving Dam fusion expression tissue-specifically. We obtained meaningful transcriptional footprints in line with RNA-seq studies in whole animals or specific tissues. To challenge the sensitivity of RAPID and demonstrate its utility to determine novel tissue-specific transcriptional profiles, we determined the transcriptional footprints of the pair of XXX neuroendocrine cells, representing 0.2% of the somatic cell content of the animals. We identified 2362 candidate genes with putatively active transcription in XXX cells, among which the few known markers for these cells. Using transcriptional reporters for a subset of new hits, we confirmed that the majority of them were expressed in XXX and identified novel XXX-specific markers. Taken together, our work establishes RAPID as a valid method for the determination of polymerase footprints in specific tissues of C. elegans without the need for cell sorting or RNA tagging.

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“…We purified ribosome-associated mRNAs and sequenced cDNA libraries to obtain translational profiles from fourth larval stage animals across three major cell types—neurons, intestinal cells, and body wall muscle cells—and validated the reproducibility and specificity of our procedure (Figures 1B and S1C-D; for detailed protocol see (Gracida and Calarco, 2017). We identified hundreds of tissueenriched mRNAs in these cell types (Figure 1C; Table S1).…”

Section: Resultsmentioning

confidence: 79%

“…Worm lysis and immunoprecipitations were performed as previously described (Heiman et al, 2014). Detailed protocol in (Gracida and Calarco, 2017). RNA was extracted using TRI reagent (Sigma T9424).…”

Section: Methodsmentioning

confidence: 99%

An Elongin-Cullin-SOCS Box Complex Regulates Stress-Induced Serotonergic Neuromodulation

et al. 2017

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Summary Neuromodulatory cells transduce environmental information into long lasting behavioral responses. However, the mechanisms governing how neuronal cells influence behavioral plasticity are difficult to characterize. Here, we adapted the Translating Ribosome Affinity Purification (TRAP) approach in C. elegans to profile ribosome-associated mRNAs from three major tissues and the neuromodulatory dopaminergic and serotonergic cells. We identified elc-2, an Elongin C ortholog, specifically expressed in stress-sensing ADF serotonergic sensory neurons, and found that it plays a role in mediating a long-lasting change in serotonin-dependent feeding behavior induced by heat stress. We demonstrate that ELC-2 and the von Hippel-Lindau protein VHL-1, components of an Elongin-CullinSOCS-box (ECS) E3 ubiquitin ligase, modulate this behavior after experiencing stress. Also, heat stress induces a transient redistribution of ELC-2, becoming more nuclearly enriched. Together, our results demonstrate dynamic regulation of an E3 ligase, and a role for an ECS complex in neuromodulation and control of lasting behavioral states.

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Cell type-specific transcriptome profiling in C. elegans using the Translating Ribosome Affinity Purification technique

Cited by 26 publications

References 33 publications

Transcriptome analysis of adult Caenorhabditis elegans cells reveals tissue-specific gene and isoform expression

Transcriptome analysis of adult Caenorhabditis elegans cells reveals tissue-specific gene and isoform expression

Tissue-Specific Transcription Footprinting Using RNA PoI DamID (RAPID) in Caenorhabditis elegans

An Elongin-Cullin-SOCS Box Complex Regulates Stress-Induced Serotonergic Neuromodulation

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