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1.
BMC Biol ; 13: 4, 2015 Jan 20.
Artículo en Inglés | MEDLINE | ID: mdl-25601023

RESUMEN

BACKGROUND: Tissue-specific RNA plasticity broadly impacts the development, tissue identity and adaptability of all organisms, but changes in composition, expression levels and its impact on gene regulation in different somatic tissues are largely unknown. Here we developed a new method, polyA-tagging and sequencing (PAT-Seq) to isolate high-quality tissue-specific mRNA from Caenorhabditis elegans intestine, pharynx and body muscle tissues and study changes in their tissue-specific transcriptomes and 3'UTRomes. RESULTS: We have identified thousands of novel genes and isoforms differentially expressed between these three tissues. The intestine transcriptome is expansive, expressing over 30% of C. elegans mRNAs, while muscle transcriptomes are smaller but contain characteristic unique gene signatures. Active promoter regions in all three tissues reveal both known and novel enriched tissue-specific elements, along with putative transcription factors, suggesting novel tissue-specific modes of transcription initiation. We have precisely mapped approximately 20,000 tissue-specific polyadenylation sites and discovered that about 30% of transcripts in somatic cells use alternative polyadenylation in a tissue-specific manner, with their 3'UTR isoforms significantly enriched with microRNA targets. CONCLUSIONS: For the first time, PAT-Seq allowed us to directly study tissue specific gene expression changes in an in vivo setting and compare these changes between three somatic tissues from the same organism at single-base resolution within the same experiment. We pinpoint precise tissue-specific transcriptome rearrangements and for the first time link tissue-specific alternative polyadenylation to miRNA regulation, suggesting novel and unexplored tissue-specific post-transcriptional regulatory networks in somatic cells.


Asunto(s)
Caenorhabditis elegans/genética , Mucosa Intestinal/metabolismo , Músculos/metabolismo , Poliadenilación/genética , Análisis de Secuencia de ARN/métodos , Regiones no Traducidas 3'/genética , Animales , Bases de Datos Genéticas , Regulación de la Expresión Génica , MicroARNs/genética , MicroARNs/metabolismo , Especificidad de Órganos/genética , Faringe/metabolismo , Poli A/metabolismo , Regiones Promotoras Genéticas/genética , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , ARN Mensajero/genética , ARN Mensajero/aislamiento & purificación , ARN Mensajero/metabolismo , Reproducibilidad de los Resultados , Transcriptoma/genética
2.
Life Sci Alliance ; 7(9)2024 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-38886018

RESUMEN

Protein translation initiation is a conserved process involving many proteins acting in concert. The 13 subunit eukaryotic initiation factor 3 (eIF3) complex is essential for assembly of the pre-initiation complex that scans mRNA and positions ribosome at the initiation codon. We previously reported that a gain-of-function (gf) mutation affecting the G subunit of the Caenorhabditis elegans eIF3 complex, eif-3.g(gf), selectively modulates protein translation in the ventral cord cholinergic motor neurons. Here, through unbiased genetic suppressor screening, we identified that the gene lin-66 mediates eif-3.g(gf)-dependent protein translation in motor neurons. LIN-66 is composed largely of low-complexity amino acid sequences with unknown functional domains. We combined bioinformatics analysis with in vivo functional dissection and identified a cold-shock domain in LIN-66 critical for its function. In cholinergic motor neurons, LIN-66 shows a close association with EIF-3.G in the cytoplasm. The low-complexity amino acid sequences of LIN-66 modulate its subcellular pattern. As cold-shock domains function broadly in RNA regulation, we propose that LIN-66 mediates stimulus-dependent protein translation by facilitating the interaction of mRNAs with EIF-3.G.


Asunto(s)
Proteínas de Caenorhabditis elegans , Caenorhabditis elegans , Factor 3 de Iniciación Eucariótica , Neuronas Motoras , Biosíntesis de Proteínas , Animales , Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/genética , Factor 3 de Iniciación Eucariótica/metabolismo , Factor 3 de Iniciación Eucariótica/genética , Neuronas Motoras/metabolismo , Mutación , ARN Mensajero/metabolismo , ARN Mensajero/genética , Secuencia de Aminoácidos , Respuesta al Choque por Frío , Dominios Proteicos
3.
STAR Protoc ; 4(1): 101959, 2023 03 17.
Artículo en Inglés | MEDLINE | ID: mdl-36566382

RESUMEN

The single-end enhanced cross-linking immunoprecipitation (seCLIP) method is well suited for efficient and unbiased transcriptome-wide interrogation of RNA-binding protein (RBP) interaction sites. Here, we provide a protocol for executing cell-specific seCLIP for any desired RBP in Caenorhabditis elegans. We begin with steps and recommendations for transgene construction and Cas9-mediated chromosomal integration. We provide detailed procedures for isolation of RBP-associated RNA fragments, subsequent library preparation, and sequencing. We further discuss best practices for data analysis, interpretation of results, and troubleshooting. For complete details on the use and execution of this protocol, please refer to Blazie et al. (2021).1.


Asunto(s)
Caenorhabditis elegans , Proteínas de Unión al ARN , Animales , Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , Sitios de Unión , Inmunoprecipitación , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismo , Transcriptoma
4.
Elife ; 102021 07 29.
Artículo en Inglés | MEDLINE | ID: mdl-34323215

RESUMEN

The translation initiation complex eIF3 imparts specialized functions to regulate protein expression. However, understanding of eIF3 activities in neurons remains limited despite widespread dysregulation of eIF3 subunits in neurological disorders. Here, we report a selective role of the C. elegans RNA-binding subunit EIF-3.G in shaping the neuronal protein landscape. We identify a missense mutation in the conserved Zinc-Finger (ZF) of EIF-3.G that acts in a gain-of-function manner to dampen neuronal hyperexcitation. Using neuron-type-specific seCLIP, we systematically mapped EIF-3.G-mRNA interactions and identified EIF-3.G occupancy on GC-rich 5'UTRs of a select set of mRNAs enriched in activity-dependent functions. We demonstrate that the ZF mutation in EIF-3.G alters translation in a 5'UTR-dependent manner. Our study reveals an in vivo mechanism for eIF3 in governing neuronal protein levels to control neuronal activity states and offers insights into how eIF3 dysregulation contributes to neurological disorders.


Asunto(s)
Proteínas de Caenorhabditis elegans/genética , Caenorhabditis elegans/fisiología , Factor 3 de Iniciación Eucariótica/genética , Neuronas/fisiología , Biosíntesis de Proteínas , ARN de Helminto/biosíntesis , ARN Mensajero/biosíntesis , Animales , Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Factor 3 de Iniciación Eucariótica/metabolismo
5.
Mol Biochem Parasitol ; 237: 111276, 2020 05.
Artículo en Inglés | MEDLINE | ID: mdl-32268182

RESUMEN

The ACC-1 family of cys-loop receptors are ligand-gated chloride channels sensitive to acetylcholine (ACh), and are only present in invertebrates. Studies of this family of inhibitory receptors has provided insight into how they bind and respond to ACh in a manner vastly different from nicotinic acetylcholine receptors and appear to be present in tissues that are relevant to anthelmintic action. Here, we have identified two members of the ACC-1 family from the parasitic nematode Haemonchus contortus, Hco-LGC-46 and Hco-ACC-4. Hco-LGC-46 is an ACC subunit that has never been previously expressed and pharmacologically characterized. We found that Hco-LGC-46 when expressed in Xenopus laevis oocytes forms a functional homomeric channel that is responsive to the cholinergic agonists ACh and methylcholine. hco-lgc-46 expressed in a C. elegans lgc-46 null strain (ok2900) suppressed hypersensitivity to aldicarb in a manner similar to cel-lgc-46. It was also found that Hco-LGC-46 assembles with Hco-ACC-1 and produces a receptor that is over 5-fold more sensitive to ACh and responds to the cholinergic agonists methycholine and carbachol. In contrast, the co-expression of Hco-LGC-46 with Hco-ACC-4 resulted in non-functional channels in oocytes. Hco-ACC-4 also appears to form heteromeric channels with a previously characterized subunit, Hco-ACC-2. Co-expression of Hco-ACC-4 with Hco-ACC-2 resulted in a functional heteromeric channel with an EC50 value similar to that of the Hco-ACC-2 homomeric channel. However, the maximum currents generated in the ACC-4/ACC-2 channel were significantly (p < 0.005) lower than those from the ACC-2 homomeric channel. Overall, this is the first report confirming that lgc-46 encodes an acetylcholine-gated chloride channel which when co-expressed with acc-4 results in reduced receptor function or trafficking in oocytes.


Asunto(s)
Acetilcolina/metabolismo , Canales de Cloruro/química , Receptores de Canales Iónicos con Asa de Cisteína Activados por Ligando/química , Haemonchus/metabolismo , Proteínas del Helminto/química , Acetilcolina/farmacología , Potenciales de Acción/efectos de los fármacos , Potenciales de Acción/fisiología , Aldicarb/farmacología , Secuencia de Aminoácidos , Animales , Antihelmínticos/farmacología , Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , Carbacol/metabolismo , Carbacol/farmacología , Canales de Cloruro/genética , Canales de Cloruro/aislamiento & purificación , Canales de Cloruro/metabolismo , Colina/análogos & derivados , Colina/metabolismo , Colina/farmacología , Clonación Molecular , Receptores de Canales Iónicos con Asa de Cisteína Activados por Ligando/genética , Receptores de Canales Iónicos con Asa de Cisteína Activados por Ligando/aislamiento & purificación , Receptores de Canales Iónicos con Asa de Cisteína Activados por Ligando/metabolismo , Expresión Génica , Vectores Genéticos/química , Vectores Genéticos/metabolismo , Haemonchus/genética , Proteínas del Helminto/genética , Proteínas del Helminto/aislamiento & purificación , Proteínas del Helminto/metabolismo , Modelos Moleculares , Oocitos/citología , Oocitos/metabolismo , Estructura Secundaria de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Xenopus laevis/genética , Xenopus laevis/metabolismo
6.
Curr Biol ; 30(5): 865-876.e7, 2020 03 09.
Artículo en Inglés | MEDLINE | ID: mdl-31983639

RESUMEN

mRNA decay factors regulate mRNA turnover by recruiting non-translating mRNAs and targeting them for translational repression and mRNA degradation. How mRNA decay pathways regulate cellular function in vivo with specificity is poorly understood. Here, we show that C. elegans mRNA decay factors, including the translational repressors CAR-1/LSM14 and CGH-1/DDX6, and the decapping enzymes DCAP-1/DCP1, function in neurons to differentially regulate axon development, maintenance, and regrowth following injury. In neuronal cell bodies, CAR-1 fully colocalizes with CGH-1 and partially colocalizes with DCAP-1, suggesting that mRNA decay components form at least two types of cytoplasmic granules. Following axon injury in adult neurons, loss of CAR-1 or CGH-1 results in increased axon regrowth and growth cone formation, whereas loss of DCAP-1 or DCAP-2 results in reduced regrowth. To determine how CAR-1 inhibits regrowth, we analyzed mRNAs bound to pan-neuronally expressed GFP::CAR-1 using a crosslinking and immunoprecipitation-based approach. Among the putative mRNA targets of CAR-1, we characterized the roles of micu-1, a regulator of the mitochondrial calcium uniporter MCU-1, in axon injury. We show that loss of car-1 results increased MICU-1 protein levels, and that enhanced axon regrowth in car-1 mutants is dependent on micu-1 and mcu-1. Moreover, axon injury induces transient calcium influx into axonal mitochondria, dependent on MCU-1. In car-1 loss-of-function mutants and in micu-1 overexpressing animals, the axonal mitochondrial calcium influx is more sustained, which likely underlies enhanced axon regrowth. Our data uncover a novel pathway that controls axon regrowth through axonal mitochondrial calcium uptake.


Asunto(s)
Axones/fisiología , Proteínas de Caenorhabditis elegans/genética , Caenorhabditis elegans/fisiología , Calcio/metabolismo , Endorribonucleasas/genética , Regeneración Nerviosa/genética , ARN Nucleotidiltransferasas/genética , Proteínas de Unión al ARN/genética , Animales , Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Endorribonucleasas/metabolismo , Dinámicas Mitocondriales , ARN Nucleotidiltransferasas/metabolismo , Estabilidad del ARN , ARN Mensajero/metabolismo , Proteínas de Unión al ARN/metabolismo
7.
Bioinformatics ; 24(22): 2622-7, 2008 Nov 15.
Artículo en Inglés | MEDLINE | ID: mdl-18786976

RESUMEN

UNLABELLED: Prokaryotic protein-protein interactions are underrepresented in currently available databases. Here, we describe a 'gold standard' dataset (MPI-LIT) focusing on microbial binary protein-protein interactions and associated experimental evidence that we have manually curated from 813 abstracts and full texts that were selected from an initial set of 36 852 abstracts. The MPI-LIT dataset comprises 1237 experimental descriptions that describe a non-redundant set of 746 interactions of which 659 (88%) are not reported in public databases. To estimate the curation quality, we compared our dataset with a union of microbial interaction data from IntAct, DIP, BIND and MINT. Among common abstracts, we achieve a sensitivity of up to 66% for interactions and 75% for experimental methods. Compared with these other datasets, MPI-LIT has the lowest fraction of interaction experiments per abstract (0.9) and the highest coverage of strains (92) and scientific articles (813). We compared methods that evaluate functional interactions among proteins (such as genomic context or co-expression) which are implemented in the STRING database. Most of these methods discriminate well between functionally relevant protein interactions (MPI-LIT) and high-throughput data. AVAILABILITY: http://www.jcvi.org/mpidb/interaction.php?dbsource=MPI-LIT. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.


Asunto(s)
Proteínas Bacterianas/metabolismo , Biología Computacional , Bases de Datos de Proteínas , Unión Proteica , Mapeo de Interacción de Proteínas
8.
ACS Chem Neurosci ; 9(8): 1963-1974, 2018 08 15.
Artículo en Inglés | MEDLINE | ID: mdl-29432681

RESUMEN

Synaptic transmission is central to nervous system function. Chemical and genetic screens are valuable approaches to probe synaptic mechanisms in living animals. The nematode Caenorhabditis elegans is a prime system to apply these methods to discover genes and dissect the cellular pathways underlying neurotransmission. Here, we review key approaches to understand neurotransmission and the action of psychiatric drugs in C. elegans. We start with early studies on cholinergic excitatory signaling at the neuromuscular junction, and move into mechanisms mediated by biogenic amines. Finally, we discuss emerging work toward understanding the mechanisms driving synaptic plasticity with a focus on regulation of protein translation.


Asunto(s)
Neurotransmisores/genética , Neurotransmisores/metabolismo , Psicotrópicos/farmacología , Transmisión Sináptica/efectos de los fármacos , Transmisión Sináptica/fisiología , Animales , Animales Modificados Genéticamente , Caenorhabditis elegans , Descubrimiento de Drogas , Plasticidad Neuronal/efectos de los fármacos , Plasticidad Neuronal/fisiología , Biosíntesis de Proteínas/efectos de los fármacos
9.
Genetics ; 206(2): 757-774, 2017 06.
Artículo en Inglés | MEDLINE | ID: mdl-28348061

RESUMEN

mRNA expression dynamics promote and maintain the identity of somatic tissues in living organisms; however, their impact in post-transcriptional gene regulation in these processes is not fully understood. Here, we applied the PAT-Seq approach to systematically isolate, sequence, and map tissue-specific mRNA from five highly studied Caenorhabditis elegans somatic tissues: GABAergic and NMDA neurons, arcade and intestinal valve cells, seam cells, and hypodermal tissues, and studied their mRNA expression dynamics. The integration of these datasets with previously profiled transcriptomes of intestine, pharynx, and body muscle tissues, precisely assigns tissue-specific expression dynamics for 60% of all annotated C. elegans protein-coding genes, providing an important resource for the scientific community. The mapping of 15,956 unique high-quality tissue-specific polyA sites in all eight somatic tissues reveals extensive tissue-specific 3'untranslated region (3'UTR) isoform switching through alternative polyadenylation (APA) . Almost all ubiquitously transcribed genes use APA and harbor miRNA targets in their 3'UTRs, which are commonly lost in a tissue-specific manner, suggesting widespread usage of post-transcriptional gene regulation modulated through APA to fine tune tissue-specific protein expression. Within this pool, the human disease gene C. elegans orthologs rack-1 and tct-1 use APA to switch to shorter 3'UTR isoforms in order to evade miRNA regulation in the body muscle tissue, resulting in increased protein expression needed for proper body muscle function. Our results highlight a major positive regulatory role for APA, allowing genes to counteract miRNA regulation on a tissue-specific basis.


Asunto(s)
Caenorhabditis elegans/genética , MicroARNs/biosíntesis , Poliadenilación/genética , Regiones no Traducidas 3' , Animales , Caenorhabditis elegans/crecimiento & desarrollo , Proteínas de Caenorhabditis elegans/genética , Modelos Animales de Enfermedad , Regulación del Desarrollo de la Expresión Génica , Humanos , MicroARNs/genética , Especificidad de Órganos/genética , Poli A/genética , Receptores Citoplasmáticos y Nucleares/genética
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