Establishment of a tissue-specific RNAi system in C. elegans

The pattern of autofluorescence in the two free-living namatodes Rhabditis dolichura and Caenorhabditis compared. In C. elegans, during later embryogenesis cells develop a typical bluish autofluorescence as illumination, while in Rh. dolichura a strong already present in the unfertilized egg. Using a new,

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A Modified TurboID Approach Identifies Tissue-Specific Centriolar Components In C. elegans

10.1101/2021.12.20.473533 ◽

2021 ◽

Author(s):

Elisabeth Holzer ◽

Cornelia Rumpf-Kienzl ◽

Sebastian Falk ◽

Alexander Dammermann

Keyword(s):

Protein Interactions ◽

Affinity Purification ◽

Experimental Models ◽

Protein Protein Interactions ◽

Tissue Specific ◽

C Elegans ◽

Early Embryos ◽

Somatic Tissues ◽

Specific Variation ◽

Labeling Method

Proximity-dependent labeling approaches such as BioID have been a great boon to studies of protein-protein interactions in the context of cytoskeletal structures such as centrosomes which are poorly amenable to traditional biochemical approaches like immunoprecipitation and tandem affinity purification. Yet, these methods have so far not been applied extensively to invertebrate experimental models such as C. elegans given the long labeling times required for the original promiscuous biotin ligase variant BirA*. Here, we show that the recently developed variant TurboID successfully probes the interactomes of both stably associated (SPD-5) and dynamically localized (PLK-1) centrosomal components. We further develop an indirect proximity labeling method employing a GFP nanobody- TurboID fusion, which allows the identification of protein interactors in a tissue-specific manner in the context of the whole animal. Critically, this approach utilizes available endogenous GFP fusions, avoiding the need to generate multiple additional strains for each target protein and the potential complications associated with overexpressing the protein from transgenes. Using this method, we identify homologs of two highly conserved centriolar components, Cep97 and Bld10/Cep135, which are present in various somatic tissues of the worm. Surprisingly, neither protein is expressed in early embryos, likely explaining why these proteins have escaped attention until now. Our work expands the experimental repertoire for C. elegans and opens the door for further studies of tissue-specific variation in centrosome architecture.

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

Genetics ◽

10.1534/genetics.120.303774 ◽

2020 ◽

Vol 216 (4) ◽

pp. 931-945 ◽

Cited By ~ 1

Author(s):

Georgina Gómez-Saldivar ◽

Jaime Osuna-Luque ◽

Jennifer I. Semple ◽

Dominique A. Glauser ◽

Sophie Jarriault ◽

...

Keyword(s):

Caenorhabditis Elegans ◽

Rna Polymerase ◽

Cell Types ◽

Neuroendocrine Cells ◽

Cell Physiology ◽

Specific Gene ◽

Cell Content ◽

Tissue Specific ◽

C Elegans ◽

Active Transcription

Differential gene expression across cell types underlies development and cell physiology in multicellular organisms. Caenorhabditis elegans is a powerful, extensively used model to address these biological questions. A remaining bottleneck relates 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 RNA Polymerase DamID (RAPID) approach, in which the Dam methyltransferase is fused to a ubiquitous RNA polymerase subunit 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, in sorted embryonic blastomeres and in different tissues from intact young adults by driving tissue-specific Dam fusion expression. We obtained meaningful transcriptional footprints in line with RNA-sequencing (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 3901 candidate genes with putatively active transcription in XXX cells, including the few previously 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 cells and identified novel XXX-specific markers. Taken together, our work establishes RAPID as a valid method for the determination of RNA polymerase footprints in specific tissues of C. elegans without the need for cell sorting or RNA tagging.

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Microtubule-dependent ribosome localization in C. elegans neurons

eLife ◽

10.7554/elife.26376 ◽

2017 ◽

Vol 6 ◽

Cited By ~ 16

Author(s):

Kentaro Noma ◽

Alexandr Goncharov ◽

Mark H Ellisman ◽

Yishi Jin

Keyword(s):

Cell Biology ◽

Neuronal Development ◽

Ultrastructural Level ◽

Synthesis Methods ◽

Tissue Specific ◽

C Elegans ◽

Multicellular Organisms ◽

Axonal Trafficking ◽

Insight Into

Subcellular localization of ribosomes defines the location and capacity for protein synthesis. Methods for in vivo visualizing ribosomes in multicellular organisms are desirable in mechanistic investigations of the cell biology of ribosome dynamics. Here, we developed an approach using split GFP for tissue-specific visualization of ribosomes in Caenorhabditis elegans. Labeled ribosomes are detected as fluorescent puncta in the axons and synaptic terminals of specific neuron types, correlating with ribosome distribution at the ultrastructural level. We found that axonal ribosomes change localization during neuronal development and after axonal injury. By examining mutants affecting axonal trafficking and performing a forward genetic screen, we showed that the microtubule cytoskeleton and the JIP3 protein UNC-16 exert distinct effects on localization of axonal and somatic ribosomes. Our data demonstrate the utility of tissue-specific visualization of ribosomes in vivo, and provide insight into the mechanisms of active regulation of ribosome localization in neurons.

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Inducible Systemic RNA Silencing in Caenorhabditis elegans

Molecular Biology of the Cell ◽

10.1091/mbc.e03-01-0858 ◽

2003 ◽

Vol 14 (7) ◽

pp. 2972-2983 ◽

Cited By ~ 86

Author(s):

Lisa Timmons ◽

Hiroaki Tabara ◽

Craig C. Mello ◽

Andrew Z. Fire

Keyword(s):

Rna Interference ◽

Caenorhabditis Elegans ◽

Rna Silencing ◽

Molecular Mechanisms ◽

Normal Animal ◽

Cell Types ◽

Animal Cells ◽

Tissue Specific ◽

C Elegans ◽

Systemic Rna

Introduction of double-stranded RNA (dsRNA) can elicit a gene-specific RNA interference response in a variety of organisms and cell types. In many cases, this response has a systemic character in that silencing of gene expression is observed in cells distal from the site of dsRNA delivery. The molecular mechanisms underlying the mobile nature of RNA silencing are unknown. For example, although cellular entry of dsRNA is possible, cellular exit of dsRNA from normal animal cells has not been directly observed. We provide evidence that transgenic strains of Caenorhabditis elegans transcribing dsRNA from a tissue-specific promoter do not exhibit comprehensive systemic RNA interference phenotypes. In these same animals, modifications of environmental conditions can result in more robust systemic RNA silencing. Additionally, we find that genetic mutations can influence the systemic character of RNA silencing in C. elegans and can separate mechanisms underlying systemic RNA silencing into tissue-specific components. These data suggest that trafficking of RNA silencing signals in C. elegans is regulated by specific physiological and genetic factors.

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Tissue specific response to DNA damage: C. elegans as role model

DNA Repair ◽

10.1016/j.dnarep.2015.04.025 ◽

2015 ◽

Vol 32 ◽

pp. 141-148 ◽

Cited By ~ 24

Author(s):

Hannes Lans ◽

Wim Vermeulen

Keyword(s):

Dna Damage ◽

Role Model ◽

Specific Response ◽

Tissue Specific ◽

C Elegans

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The spatial dynamics of tissue-specific promoters during C. elegans development

Genes & Development ◽

10.1101/gad.559610 ◽

2010 ◽

Vol 24 (8) ◽

pp. 766-782 ◽

Cited By ~ 142

Author(s):

P. Meister ◽

B. D. Towbin ◽

B. L. Pike ◽

A. Ponti ◽

S. M. Gasser

Keyword(s):

Spatial Dynamics ◽

Tissue Specific ◽

C Elegans

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Tissue-Specific Activities of C. elegans DAF-16 in the Regulation of Lifespan

Cell ◽

10.1016/s0092-8674(03)00889-4 ◽

2003 ◽

Vol 115 (4) ◽

pp. 489-502 ◽

Cited By ~ 511

Author(s):

Nataliya Libina ◽

Jennifer R. Berman ◽

Cynthia Kenyon

Keyword(s):

Tissue Specific ◽

C Elegans ◽

Specific Activities

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New strains for tissue-specific RNAi studies in Caenorhabditis elegans

10.1101/283325 ◽

2018 ◽

Author(s):

Jason S. Watts ◽

Henry F. Harrison ◽

Shizue Omi ◽

Quentin Guenthers ◽

James Dalelio ◽

...

Keyword(s):

Fatty Acid ◽

Caenorhabditis Elegans ◽

Gene Function ◽

Target Genes ◽

Resistant Mutant ◽

Knockout Mutant ◽

Tissue Specific ◽

Double Stranded Rna ◽

C Elegans ◽

Insight Into

AbstractRNA interference is a powerful tool for dissecting gene function. In Caenorhabditis elegans, ingestion of double stranded RNA causes strong, systemic knockdown of target genes. Further insight into gene function can be revealed by tissue-specific RNAi techniques. Currently available tissue-specific C. elegans strains rely on rescue of RNAi function in a desired tissue or cell in an otherwise RNAi deficient genetic background. We attempted to assess the contribution of specific tissues to polyunsaturated fatty acid (PUFA) synthesis using currently available tissue-specific RNAi strains. We discovered that rde-1 (ne219), a commonly used RNAi-resistant mutant strain, retains considerable RNAi capacity against RNAi directed at PUFA synthesis genes. By measuring changes in the fatty acid products of the desaturase enzymes that synthesize PUFAs, we found that the before mentioned strain, rde-1 (ne219) and the reported germline only RNAi strain, rrf-1 (pk1417) are not appropriate genetic backgrounds for tissue-specific RNAi experiments. However, the knockout mutant rde-1 (ne300) was strongly resistant to dsRNA induced RNAi, and thus is more appropriate for construction of a robust tissue-specific RNAi strains. Using newly constructed strains in the rde-1(null) background, we found considerable desaturase activity in intestinal, epidermal, and germline tissues, but not in muscle. The RNAi-specific strains reported in this study will be useful tools for C. elegans researchers studying a variety of biological processes.

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