RESUMEN
Comprised of only 20 cells, the Caenorhabditis elegans intestine is the nexus of many life-supporting functions, including digestion, metabolism, aging, immunity, and environmental response. Critical interactions between the C. elegans host and its environment converge within the intestine, where gut microbiota concentrate. Therefore, the ability to isolate intestine tissue away from the rest of the worm is necessary to assess intestine-specific processes. This protocol describes a method for hand dissecting adult C. elegans intestines. The procedure can be performed in fluorescently labeled strains for ease or training purposes. Once the technique is perfected, intestines can be collected from unlabeled worms of any genotype. This microdissection approach allows for the simultaneous capture of host intestinal tissue and gut microbiota, a benefit to many microbiome studies. As such, downstream applications for the intestinal preparations generated by this protocol can include but are not limited to RNA isolation from intestinal cells and DNA isolation from captured microbiota. Overall, hand dissection of C. elegans intestines affords a simple and robust method to investigate critical aspects of intestine biology.
Asunto(s)
Proteínas de Caenorhabditis elegans , Microbioma Gastrointestinal , Microbiota , Animales , Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/genética , Intestinos/fisiología , Intestinos/cirugía , ARNRESUMEN
Aneuploidy, the incorrect number of whole chromosomes, is a common feature of tumors that contributes to their initiation and evolution. Preventing aneuploidy requires properly functioning kinetochores, which are large protein complexes assembled on centromeric DNA that link mitotic chromosomes to dynamic spindle microtubules and facilitate chromosome segregation. The kinetochore leverages at least two mechanisms to prevent aneuploidy: error correction and the spindle assembly checkpoint (SAC). BubR1, a factor involved in both processes, was identified as a cancer dependency and therapeutic target in multiple tumor types; however, it remains unclear what specific oncogenic pressures drive this enhanced dependency on BubR1 and whether it arises from BubR1's regulation of the SAC or error-correction pathways. Here, we use a genetically controlled transformation model and glioblastoma tumor isolates to show that constitutive signaling by RAS or MAPK is necessary for cancer-specific BubR1 vulnerability. The MAPK pathway enzymatically hyperstimulates a network of kinetochore kinases that compromises chromosome segregation, rendering cells more dependent on two BubR1 activities: counteracting excessive kinetochore-microtubule turnover for error correction and maintaining the SAC. This work expands our understanding of how chromosome segregation adapts to different cellular states and reveals an oncogenic trigger of a cancer-specific defect.
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Neoplasias , Proteínas Serina-Treonina Quinasas , Aneuploidia , Carcinogénesis/metabolismo , Proteínas de Ciclo Celular/metabolismo , Segregación Cromosómica , Humanos , Cinetocoros/metabolismo , Microtúbulos/metabolismo , Mitosis/genética , Neoplasias/metabolismo , Proteínas Serina-Treonina Quinasas/genética , Huso Acromático/metabolismoRESUMEN
Cells spatially organize their molecular components to carry out fundamental biological processes and guide proper development. The spatial organization of RNA within the cell can both promote and result from gene expression regulatory control. Recent studies have demonstrated diverse associations between RNA spatial patterning and translation regulatory control. One form of patterning, compartmentalization in biomolecular condensates, has been of particular interest. Generally, transcripts associated with cytoplasmic biomolecular condensates-such as germ granules, stress granules, and P-bodies-are linked with low translational status. However, recent studies have identified new biomolecular condensates with diverse roles associated with active translation. This review outlines RNA compartmentalization in various condensates that occur in association with repressed or active translational states, highlights recent findings in well-studied condensates, and explores novel condensate behaviors.
RESUMEN
Low complexity domains (LCDs) in proteins are regions predominantly composed of a small subset of the possible amino acids. LCDs are involved in a variety of normal and pathological processes across all domains of life. Existing methods define LCDs using information-theoretical complexity thresholds, sequence alignment with repetitive regions, or statistical overrepresentation of amino acids relative to whole-proteome frequencies. While these methods have proven valuable, they are all indirectly quantifying amino acid composition, which is the fundamental and biologically-relevant feature related to protein sequence complexity. Here, we present a new computational tool, LCD-Composer, that directly identifies LCDs based on amino acid composition and linear amino acid dispersion. Using LCD-Composer's default parameters, we identified simple LCDs across all organisms available through UniProt and provide the resulting data in an accessible form as a resource. Furthermore, we describe large-scale differences between organisms from different domains of life and explore organisms with extreme LCD content for different LCD classes. Finally, we illustrate the versatility and specificity achievable with LCD-Composer by identifying diverse classes of LCDs using both simple and multifaceted composition criteria. We demonstrate that the ability to dissect LCDs based on these multifaceted criteria enhances the functional mapping and classification of LCDs.
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Caenorhabditis elegans early embryos generate cell-specific transcriptomes despite lacking active transcription, thereby presenting an opportunity to study mechanisms of post-transcriptional regulatory control. We observed that some cell-specific mRNAs accumulate non-homogenously within cells, localizing to membranes, P granules (associated with progenitor germ cells in the P lineage) and P-bodies (associated with RNA processing). The subcellular distribution of transcripts differed in their dependence on 3'UTRs and RNA binding proteins, suggesting diverse regulatory mechanisms. Notably, we found strong but imperfect correlations between low translational status and P granule localization within the progenitor germ lineage. By uncoupling translation from mRNA localization, we untangled a long-standing question: Are mRNAs directed to P granules to be translationally repressed, or do they accumulate there as a consequence of this repression? We found that translational repression preceded P granule localization and could occur independently of it. Further, disruption of translation was sufficient to send homogenously distributed mRNAs to P granules. These results implicate transcriptional repression as a means to deliver essential maternal transcripts to the progenitor germ lineage for later translation.
Asunto(s)
Caenorhabditis elegans/metabolismo , Células Germinativas/metabolismo , ARN Mensajero/metabolismo , Animales , Proteínas de Caenorhabditis elegans/metabolismoRESUMEN
Plant nucleotide-binding leucine-rich repeat (NLR) immune receptors activate cell death and confer disease resistance by unknown mechanisms. We demonstrate that plant Toll/interleukin-1 receptor (TIR) domains of NLRs are enzymes capable of degrading nicotinamide adenine dinucleotide in its oxidized form (NAD+). Both cell death induction and NAD+ cleavage activity of plant TIR domains require known self-association interfaces and a putative catalytic glutamic acid that is conserved in both bacterial TIR NAD+-cleaving enzymes (NADases) and the mammalian SARM1 (sterile alpha and TIR motif containing 1) NADase. We identify a variant of cyclic adenosine diphosphate ribose as a biomarker of TIR enzymatic activity. TIR enzymatic activity is induced by pathogen recognition and functions upstream of the genes enhanced disease susceptibility 1 (EDS1) and N requirement gene 1 (NRG1), which encode regulators required for TIR immune function. Thus, plant TIR-NLR receptors require NADase function to transduce recognition of pathogens into a cell death response.
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Arabidopsis/enzimología , Arabidopsis/inmunología , Dominio Catalítico , NAD+ Nucleosidasa/química , NAD/metabolismo , Receptores Inmunológicos/química , Sustitución de Aminoácidos , Arabidopsis/microbiología , Proteínas de Arabidopsis/metabolismo , Proteínas del Dominio Armadillo/química , Biomarcadores/análisis , Biomarcadores/metabolismo , Muerte Celular , Secuencia Conservada , ADP-Ribosa Cíclica/análisis , ADP-Ribosa Cíclica/metabolismo , Proteínas del Citoesqueleto/química , Proteínas de Unión al ADN/metabolismo , Ácido Glutámico/química , Ácido Glutámico/genética , Interacciones Huésped-PatógenoRESUMEN
The two GATA transcription factors ELT-2 and ELT-7 function in the differentiation of the C. elegans intestine. ELT-2 loss causes lethality. ELT-7 loss causes no obvious phenotype but enhances the elt-2(-) intestinal phenotype. Thus, ELT-2 and ELT-7 appear partially redundant, with ELT-2 being more influential. To investigate the different regulatory roles of ELT-2 and ELT-7, we compared the transcriptional profiles of pure populations of wild-type, elt-2(-), elt-7(-), and elt-7(-); elt-2(-) double mutant L1-stage larvae. Consistent with the mutant phenotypes, loss of ELT-2 had a>25 fold greater influence on the number of significantly altered transcripts compared to the loss of ELT-7; nonetheless, the levels of numerous transcripts changed upon loss of ELT-7 in the elt-2(-) background. The quantitative responses of individual genes revealed a more complicated behaviour than simple redundancy/partial redundancy. In particular, genes expressed only in the intestine showed three distinguishable classes of response in the different mutant backgrounds. One class of genes responded as if ELT-2 is the major transcriptional activator and ELT-7 provides variable compensatory input. For a second class, transcript levels increased upon loss of ELT-2 but decreased upon further loss of ELT-7, suggesting that ELT-7 actually overcompensates for the loss of ELT-2. For a third class, transcript levels also increased upon loss of ELT-2 but remained elevated upon further loss of ELT-7, suggesting overcompensation by some other intestinal transcription factor(s). In spite of its minor loss-of-function phenotype and its limited sequence similarity to ELT-2, ELT-7 expressed under control of the elt-2 promoter is able to rescue elt-2(-) lethality. Indeed, appropriately expressed ELT-7, like appropriately expressed ELT-2, is able to replace all other core GATA factors in the C. elegans endodermal pathway. Overall, this study focuses attention on the quantitative intricacies behind apparent redundancy or partial redundancy of two related transcription factors.
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Proteínas de Caenorhabditis elegans/fisiología , Caenorhabditis elegans/genética , Endodermo/metabolismo , Factores de Transcripción GATA/fisiología , Regulación del Desarrollo de la Expresión Génica , Mucosa Intestinal/metabolismo , Animales , Caenorhabditis elegans/embriología , Caenorhabditis elegans/crecimiento & desarrollo , Proteínas de Caenorhabditis elegans/genética , Diferenciación Celular , Factores de Transcripción GATA/deficiencia , Factores de Transcripción GATA/genética , Genes de Helminto , Genes Reporteros , Estudios de Asociación Genética , Intestinos/citología , Larva , Regiones Promotoras Genéticas , Transcripción Genética , TranscriptomaRESUMEN
Detection of pathogens by plants is mediated by intracellular nucleotide-binding site leucine-rich repeat (NLR) receptor proteins. NLR proteins are defined by their stereotypical multidomain structure: an N-terminal Toll-interleukin receptor (TIR) or coiled-coil (CC) domain, a central nucleotide-binding (NB) domain, and a C-terminal leucine-rich repeat (LRR). The plant innate immune system contains a limited NLR repertoire that functions to recognize all potential pathogens. We isolated Response to the bacterial type III effector protein HopBA1 (RBA1), a gene that encodes a TIR-only protein lacking all other canonical NLR domains. RBA1 is sufficient to trigger cell death in response to HopBA1. We generated a crystal structure for HopBA1 and found that it has similarity to a class of proteins that includes esterases, the heme-binding protein ChaN, and an uncharacterized domain of Pasteurella multocida toxin. Self-association, coimmunoprecipitation with HopBA1, and function of RBA1 require two previously identified TIR-TIR dimerization interfaces. Although previously described as distinct in other TIR proteins, in RBA1 neither of these interfaces is sufficient when the other is disrupted. These data suggest that oligomerization of RBA1 is required for function. Our identification of RBA1 demonstrates that "truncated" NLRs can function as pathogen sensors, expanding our understanding of both receptor architecture and the mechanism of activation in the plant immune system.
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Proteínas de Arabidopsis/química , Arabidopsis/química , Arabidopsis/genética , Regulación de la Expresión Génica de las Plantas , Enfermedades de las Plantas/genética , Proteínas de Plantas/química , Arabidopsis/inmunología , Arabidopsis/microbiología , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/inmunología , Sitios de Unión , Muerte Celular/genética , Muerte Celular/inmunología , Cristalografía por Rayos X , Erwinia/patogenicidad , Erwinia/fisiología , Interacciones Huésped-Patógeno , Modelos Moleculares , Mutación , Enfermedades de las Plantas/inmunología , Enfermedades de las Plantas/microbiología , Inmunidad de la Planta/genética , Proteínas de Plantas/genética , Proteínas de Plantas/inmunología , Unión Proteica , Dominios y Motivos de Interacción de Proteínas , Estructura Secundaria de Proteína , Pseudomonas syringae/patogenicidad , Pseudomonas syringae/fisiología , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/inmunología , Transducción de Señal , Nicotiana/genética , Nicotiana/inmunología , Nicotiana/microbiología , Sistemas de Secreción Tipo III/genética , Sistemas de Secreción Tipo III/metabolismoRESUMEN
The ELT-2 GATA factor is the predominant transcription factor regulating gene expression in the C. elegans intestine, following endoderm specification. We comment on our previous study (Wiesenfahrt et al., 2016) that investigated how the elt-2 gene is controlled by END-1, END-3 and ELT-7, the 3 endoderm specific GATA factors that lie upstream in the regulatory hierarchy. We also discuss the unexpected result that ELT-2, if expressed sufficiently early and at sufficiently high levels, can specify the C. elegans endoderm, replacing the normal functions of END-1 and END-3.
RESUMEN
P granules are non-membrane-bound RNA-protein compartments that are involved in germline development in C. elegans. They are liquids that condense at one end of the embryo by localized phase separation, driven by gradients of polarity proteins such as the mRNA-binding protein MEX-5. To probe how polarity proteins regulate phase separation, we combined biochemistry and theoretical modeling. We reconstitute P granule-like droplets in vitro using a single protein PGL-3. By combining in vitro reconstitution with measurements of intracellular concentrations, we show that competition between PGL-3 and MEX-5 for mRNA can regulate the formation of PGL-3 droplets. Using theory, we show that, in a MEX-5 gradient, this mRNA competition mechanism can drive a gradient of P granule assembly with similar spatial and temporal characteristics to P granule assembly in vivo. We conclude that gradients of polarity proteins can position RNP granules during development by using RNA competition to regulate local phase separation.
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Caenorhabditis elegans/metabolismo , ARN Mensajero/metabolismo , Animales , Proteínas de Caenorhabditis elegans/análisis , Proteínas de Caenorhabditis elegans/metabolismo , Polaridad Celular , Embrión no Mamífero , Espacio Intracelular/química , Espacio Intracelular/metabolismo , Modelos Teóricos , Unión Proteica , Proteínas de Unión al ARN/análisis , Proteínas de Unión al ARN/metabolismoRESUMEN
During embryonic development, cells must establish fates, morphologies, and behaviors in coordination with one another to form a functional body. A prevalent hypothesis for how this coordination is achieved is that each cell's fate and behavior is determined by a defined mixture of RNAs. Only recently has it become possible to measure the full suite of transcripts in a single cell. Here we quantify genome-wide mRNA abundance in each cell of the Caenorhabditis elegans embryo up to the 16-cell stage. We describe spatially dynamic expression, quantify cell-specific differential activation of the zygotic genome, and identify genes that were previously unappreciated as being critical for development. We present an interactive data visualization tool that allows broad access to our dataset. This genome-wide single-cell map of mRNA abundance, alongside the well-studied life history and fate of each cell, describes at a cellular resolution the mRNA landscape that guides development.
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Caenorhabditis elegans/embriología , Linaje de la Célula/genética , Desarrollo Embrionario/genética , Regulación del Desarrollo de la Expresión Génica/genética , Animales , Secuencia de Bases , Caenorhabditis elegans/genética , Embrión no Mamífero/citología , Perfilación de la Expresión Génica , ARN Mensajero/genética , Análisis de Secuencia de ARN , Transcriptoma/genética , Cigoto/citologíaRESUMEN
ELT-2 is the major regulator of genes involved in differentiation, maintenance and function of C. elegans intestine from the early embryo to mature adult. elt-2 responds to overexpression of the GATA transcription factors END-1 and END-3, which specify the intestine, as well as to overexpression of the two GATA factors that are normally involved in intestinal differentiation, ELT-7 and ELT-2 itself. Little is known about the molecular mechanisms underlying these interactions, how ELT-2 levels are maintained throughout development or how such systems respond to developmental perturbations. Here, we analyse elt-2 gene regulation through transgenic reporter assays, ELT-2 ChIP and characterisation of in vitro DNA-protein interactions. Our results indicate that elt-2 is controlled by three discrete regulatory regions conserved between C. elegans and C. briggsae that span >4â kb of 5' flanking sequence. These regions are superficially interchangeable but have quantitatively different enhancer properties, and their combined activities indicate inter-region synergies. Their regulatory activity is mediated by a small number of conserved TGATAA sites that are largely interchangeable and interact with different endodermal GATA factors with only modest differences in affinity. The redundant molecular mechanism that forms the elt-2 regulatory network is robust and flexible, as loss of end-3 halves ELT-2 levels in the early embryo but levels fully recover by the time of hatching. When ELT-2 is expressed under the control of end-1 regulatory elements, in addition to its own endogenous promoter, it can replace the complete set of endoderm-specific GATA factors: END-1, END-3, ELT-7 and (the probably non-functional) ELT-4. Thus, in addition to controlling gene expression during differentiation, ELT-2 is capable of specifying the entire C. elegans endoderm.
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Proteínas de Caenorhabditis elegans/genética , Caenorhabditis elegans/embriología , Caenorhabditis elegans/genética , Endodermo/embriología , Endodermo/metabolismo , Factores de Transcripción GATA/genética , Regulación del Desarrollo de la Expresión Génica , Región de Flanqueo 5'/genética , Animales , Secuencia de Bases , Proteínas de Caenorhabditis elegans/metabolismo , Diferenciación Celular/genética , Inmunoprecipitación de Cromatina , Secuencia Conservada , ADN/metabolismo , Factores de Transcripción GATA/metabolismo , Redes Reguladoras de Genes , Mucosa Intestinal/metabolismo , Datos de Secuencia Molecular , Regiones Promotoras Genéticas , Unión Proteica/genética , Factores de Transcripción/metabolismo , Transcripción GenéticaRESUMEN
After fertilization but prior to the onset of zygotic transcription, the C. elegans zygote cleaves asymmetrically to create the anterior AB and posterior P1 blastomeres, each of which goes on to generate distinct cell lineages. To understand how patterns of RNA inheritance and abundance arise after this first asymmetric cell division, we pooled hand-dissected AB and P1 blastomeres and performed RNA-seq. Our approach identified over 200 asymmetrically abundant mRNA transcripts. We confirmed symmetric or asymmetric abundance patterns for a subset of these transcripts using smFISH. smFISH also revealed heterogeneous subcellular patterning of the P1-enriched transcripts chs-1 and bpl-1. We screened transcripts enriched in a given blastomere for embryonic defects using RNAi. The gene neg-1 (F32D1.6) encoded an AB-enriched (anterior) transcript and was required for proper morphology of anterior tissues. In addition, analysis of the asymmetric transcripts yielded clues regarding the post-transcriptional mechanisms that control cellular mRNA abundance during asymmetric cell divisions, which are common in developing organisms.
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División Celular Asimétrica , Blastómeros/metabolismo , Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/genética , Morfogénesis , Proteínas Nucleares/metabolismo , ARN Mensajero/metabolismo , Animales , Caenorhabditis elegans/embriología , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/genética , Proteínas Nucleares/genética , ARN Mensajero/genéticaRESUMEN
Advances in sequencing technology have significantly advanced the landscape of developmental biology research. The dissection of genetic networks in model and non-model organisms has been greatly enhanced with high-throughput sequencing technologies. RNA-seq has revolutionized the ability to perform developmental biology research in organisms without a published genome sequence. Here, we describe a protocol for developmental biologists to perform RNA-seq on dissected tissue or whole embryos. We start with the isolation of RNA and generation of sequencing libraries. We further show how to interpret and analyze the large amount of sequencing data that is generated in RNA-seq. We explore the abilities to examine differential expression, gene duplication, transcript assembly, alternative splicing and SNP discovery. For the purposes of this article, we use Xenopus laevis as the model organism to discuss uses of RNA-seq in an organism without a fully annotated genome sequence.
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Genoma , Análisis de Secuencia de ARN/métodos , Xenopus laevis/genética , Animales , Biología Evolutiva/métodos , Modelos Animales , Xenopus laevis/crecimiento & desarrolloRESUMEN
Silencing at the HMR and HML loci in Saccharomyces cerevisiae requires recruitment of Sir proteins to the HML and HMR silencers. The silencers are regulatory sites flanking both loci and consisting of binding sites for the Rap1, Abf1, and ORC proteins, each of which also functions at hundreds of sites throughout the genome in processes unrelated to silencing. Interestingly, the sequence of the binding site for Rap1 at the silencers is distinct from the genome-wide binding profile of Rap1, being a weaker match to the consensus, and indeed is bound with low affinity relative to the consensus sequence. Remarkably, this low-affinity Rap1 binding site variant was conserved among silencers of the sensu stricto Saccharomyces species, maintained as a poor match to the Rap1 genome-wide consensus sequence in all of them. We tested multiple predictions about the possible role of this binding-site variant in silencing by substituting the native Rap1 binding site at the HMR-E silencer with the genome-wide consensus sequence for Rap1. Contrary to the predictions from the current models of Rap1, we found no influence of the Rap1 binding site version on the kinetics of establishing silencing, nor on the maintenance of silencing, nor the extent of silencing. We further explored implications of these findings with regard to prevention of ectopic silencing, and deduced that the selective pressure for the unprecedented conservation of this binding site variant may not be related to silencing.