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1.
Mol Cell ; 72(3): 510-524.e12, 2018 11 01.
Artigo em Inglês | MEDLINE | ID: mdl-30388412

RESUMO

Alternative splicing is crucial for diverse cellular, developmental, and pathological processes. However, the full networks of factors that control individual splicing events are not known. Here, we describe a CRISPR-based strategy for the genome-wide elucidation of pathways that control splicing and apply it to microexons with important functions in nervous system development and that are commonly misregulated in autism. Approximately 200 genes associated with functionally diverse regulatory layers and enriched in genetic links to autism control neuronal microexons. Remarkably, the widely expressed RNA binding proteins Srsf11 and Rnps1 directly, preferentially, and frequently co-activate these microexons. These factors form critical interactions with the neuronal splicing regulator Srrm4 and a bi-partite intronic splicing enhancer element to promote spliceosome formation. Our study thus presents a versatile system for the identification of entire splicing regulatory pathways and further reveals a common mechanism for the definition of neuronal microexons that is disrupted in autism.


Assuntos
Processamento Alternativo/fisiologia , Engenharia Genética/métodos , Sítios de Splice de RNA/fisiologia , Animais , Transtorno Autístico/genética , Sistemas CRISPR-Cas/genética , Linhagem Celular , Éxons/fisiologia , Humanos , Camundongos , Proteínas do Tecido Nervoso , Neurogênese , Neurônios , Precursores de RNA/fisiologia , Splicing de RNA/fisiologia , Proteínas de Ligação a RNA , Ribonucleoproteínas , Fatores de Processamento de Serina-Arginina , Spliceossomos
2.
J Virol ; 97(10): e0039623, 2023 10 31.
Artigo em Inglês | MEDLINE | ID: mdl-37706687

RESUMO

IMPORTANCE: This study highlights the crucial role RNA processing plays in regulating viral gene expression and replication. By targeting SR kinases, we identified harmine as a potent inhibitor of HIV-1 as well as coronavirus (HCoV-229E and multiple SARS-CoV-2 variants) replication. Harmine inhibits HIV-1 protein expression and reduces accumulation of HIV-1 RNAs in both cell lines and primary CD4+ T cells. Harmine also suppresses coronavirus replication post-viral entry by preferentially reducing coronavirus sub-genomic RNA accumulation. By focusing on host factors rather than viral targets, our study offers a novel approach to combating viral infections that is effective against a range of unrelated viruses. Moreover, at doses required to inhibit virus replication, harmine had limited toxicity and minimal effect on the host transcriptome. These findings support the viability of targeting host cellular processes as a means of developing broad-spectrum anti-virals.


Assuntos
Antivirais , Coronavirus , HIV-1 , Harmina , Humanos , Antivirais/farmacologia , Antivirais/uso terapêutico , Coronavirus/efeitos dos fármacos , Coronavirus/fisiologia , Infecções por Coronavirus/tratamento farmacológico , Harmina/farmacologia , Harmina/uso terapêutico , HIV-1/efeitos dos fármacos , HIV-1/fisiologia , Replicação Viral/efeitos dos fármacos
3.
Cell ; 138(5): 898-910, 2009 Sep 04.
Artigo em Inglês | MEDLINE | ID: mdl-19737518

RESUMO

Alternative splicing is a key process underlying the evolution of increased proteomic and functional complexity and is especially prevalent in the mammalian nervous system. However, the factors and mechanisms governing nervous system-specific alternative splicing are not well understood. Through a genome-wide computational and expression profiling strategy, we have identified a tissue- and vertebrate-restricted Ser/Arg (SR) repeat splicing factor, the neural-specific SR-related protein of 100 kDa (nSR100). We show that nSR100 regulates an extensive network of brain-specific alternative exons enriched in genes that function in neural cell differentiation. nSR100 acts by increasing the levels of the neural/brain-enriched polypyrimidine tract binding protein and by interacting with its target transcripts. Disruption of nSR100 prevents neural cell differentiation in cell culture and in the developing zebrafish. Our results thus reveal a critical neural-specific alternative splicing regulator, the evolution of which has contributed to increased complexity in the vertebrate nervous system.


Assuntos
Processamento Alternativo , Proteínas do Tecido Nervoso/metabolismo , Proteínas Nucleares/metabolismo , Proteínas de Ligação a RNA/metabolismo , Animais , Encéfalo/citologia , Diferenciação Celular , Linhagem Celular , Humanos , Camundongos , Proteínas do Tecido Nervoso/química , Proteínas do Tecido Nervoso/genética , Neurônios/citologia , Proteínas Nucleares/química , Proteínas de Ligação a RNA/química , Fatores de Processamento de Serina-Arginina
4.
Genome Res ; 30(12): 1766-1780, 2020 12.
Artigo em Inglês | MEDLINE | ID: mdl-33127752

RESUMO

Alternative splicing plays a major role in shaping tissue-specific transcriptomes. Among the broad tissue types present in metazoans, the central nervous system contains some of the highest levels of alternative splicing. Although many documented examples of splicing differences between broad tissue types exist, there remains much to be understood about the splicing factors and the cis sequence elements controlling tissue and neuron subtype-specific splicing patterns. By using translating ribosome affinity purification coupled with deep-sequencing (TRAP-seq) in Caenorhabditis elegans, we have obtained high coverage profiles of ribosome-associated mRNA for three broad tissue classes (nervous system, muscle, and intestine) and two neuronal subtypes (dopaminergic and serotonergic neurons). We have identified hundreds of splice junctions that exhibit distinct splicing patterns between tissue types or within the nervous system. Alternative splicing events differentially regulated between tissues are more often frame-preserving, are more highly conserved across Caenorhabditis species, and are enriched in specific cis regulatory motifs, when compared with other types of exons. By using this information, we have identified a likely mechanism of splicing repression by the RNA-binding protein UNC-75/CELF via interactions with cis elements that overlap a 5' splice site. Alternatively spliced exons also overlap more frequently with intrinsically disordered peptide regions than constitutive exons. Moreover, regulated exons are often shorter than constitutive exons but are flanked by longer intron sequences. Among these tissue-regulated exons are several highly conserved microexons <27 nt in length. Collectively, our results indicate a rich layer of tissue-specific gene regulation at the level of alternative splicing in C. elegans that parallels the evolutionary forces and constraints observed across metazoa.


Assuntos
Processamento Alternativo , Proteínas de Caenorhabditis elegans/genética , Caenorhabditis elegans/genética , Perfilação da Expressão Gênica/métodos , Proteínas de Ligação a RNA/metabolismo , Animais , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/metabolismo , Éxons , Regulação da Expressão Gênica , Sequenciamento de Nucleotídeos em Larga Escala , Intestinos/química , Músculo Esquelético/química , Sistema Nervoso/química , Especificidade de Órgãos , Análise de Sequência de RNA , Distribuição Tecidual
5.
Mol Cell ; 54(6): 946-959, 2014 Jun 19.
Artigo em Inglês | MEDLINE | ID: mdl-24910101

RESUMO

Alternative splicing is important for the development and function of the nervous system, but little is known about the differences in alternative splicing between distinct types of neurons. Furthermore, the factors that control cell-type-specific splicing and the physiological roles of these alternative isoforms are unclear. By monitoring alternative splicing at single-cell resolution in Caenorhabditis elegans, we demonstrate that splicing patterns in different neurons are often distinct and highly regulated. We identify two conserved RNA-binding proteins, UNC-75/CELF and EXC-7/Hu/ELAV, which regulate overlapping networks of splicing events in GABAergic and cholinergic neurons. We use the UNC-75 exon network to discover regulators of synaptic transmission and to identify unique roles for isoforms of UNC-64/Syntaxin, a protein required for synaptic vesicle fusion. Our results indicate that combinatorial regulation of alternative splicing in distinct neurons provides a mechanism to specialize metazoan nervous systems.


Assuntos
Processamento Alternativo/genética , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/fisiologia , Caenorhabditis elegans/genética , Neurônios Colinérgicos/citologia , Neurônios GABAérgicos/citologia , Proteínas de Ligação a RNA/fisiologia , Transmissão Sináptica/genética , Sintaxina 1/genética , Animais , Neurônios Colinérgicos/metabolismo , Neurônios GABAérgicos/metabolismo , Mutação , Sistema Nervoso/embriologia , Sistema Nervoso/crescimento & desenvolvimento , Isoformas de Proteínas/genética , Proteínas de Ligação a RNA/genética , Transmissão Sináptica/fisiologia , Vesículas Sinápticas/metabolismo
6.
PLoS Genet ; 14(4): e1007303, 2018 04.
Artigo em Inglês | MEDLINE | ID: mdl-29649217

RESUMO

UBR1 is an E3 ubiquitin ligase best known for its ability to target protein degradation by the N-end rule. The physiological functions of UBR family proteins, however, remain not fully understood. We found that the functional loss of C. elegans UBR-1 leads to a specific motor deficit: when adult animals generate reversal movements, A-class motor neurons exhibit synchronized activation, preventing body bending. This motor deficit is rescued by removing GOT-1, a transaminase that converts aspartate to glutamate. Both UBR-1 and GOT-1 are expressed and critically required in premotor interneurons of the reversal motor circuit to regulate the motor pattern. ubr-1 and got-1 mutants exhibit elevated and decreased glutamate level, respectively. These results raise an intriguing possibility that UBR proteins regulate glutamate metabolism, which is critical for neuronal development and signaling.


Assuntos
Caenorhabditis elegans/fisiologia , Ácido Glutâmico/metabolismo , Movimento , Ubiquitina-Proteína Ligases/metabolismo , Animais , Caenorhabditis elegans/enzimologia , Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans , Neurônios Motores/fisiologia , Ubiquitina-Proteína Ligases/genética
7.
Mol Cell ; 46(6): 884-92, 2012 Jun 29.
Artigo em Inglês | MEDLINE | ID: mdl-22749401

RESUMO

Alternative splicing plays a key role in the expansion of proteomic and regulatory complexity, yet the functions of the vast majority of differentially spliced exons are not known. In this study, we observe that brain and other tissue-regulated exons are significantly enriched in flexible regions of proteins that likely form conserved interaction surfaces. These proteins participate in significantly more interactions in protein-protein interaction (PPI) networks than other proteins. Using LUMIER, an automated PPI assay, we observe that approximately one-third of analyzed neural-regulated exons affect PPIs. Inclusion of these exons stimulated and repressed different partner interactions at comparable frequencies. This assay further revealed functions of individual exons, including a role for a neural-specific exon in promoting an interaction between Bridging Integrator 1 (Bin1)/Amphiphysin II and Dynamin 2 (Dnm2) that facilitates endocytosis. Collectively, our results provide evidence that regulated alternative exons frequently remodel interactions to establish tissue-dependent PPI networks.


Assuntos
Processamento Alternativo , Mapas de Interação de Proteínas , Proteínas/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Sítios de Ligação , Células Cultivadas , Dinamina II/genética , Dinamina II/metabolismo , Éxons , Células HEK293 , Humanos , Luciferases de Renilla/genética , Luciferases de Renilla/metabolismo , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Proteínas/genética , Proteômica , Proteínas Supressoras de Tumor/genética , Proteínas Supressoras de Tumor/metabolismo
8.
Methods ; 126: 130-137, 2017 08 15.
Artigo em Inglês | MEDLINE | ID: mdl-28648677

RESUMO

Organs and specific cell types execute specialized functions in multicellular organisms, in large part through customized gene expression signatures. Thus, profiling the transcriptomes of specific cell and tissue types remains an important tool for understanding how cells become specialized. Methodological approaches to detect gene expression differences have utilized samples from whole animals, dissected tissues, and more recently single cells. Despite these advances, there is still a challenge and a need in most laboratories to implement less invasive yet powerful cell-type specific transcriptome profiling methods. Here, we describe the use of the Translating Ribosome Affinity Purification (TRAP) method for C. elegans to detect cell type-specific gene expression patterns at the level of translating mRNAs. In TRAP, a ribosomal protein is fused to a tag (GFP) and is expressed under cell type-specific promoters to mark genetically defined cell types in vivo. Affinity purification of lysates of animals expressing the tag enriches for ribosome-associated mRNAs of the targeted tissue. The purified mRNAs are used for making cDNA libraries subjected to high-throughput sequencing to obtain genome-wide profiles of transcripts from the targeted cell type. The ease of exposing C. elegans to diverse stimuli, coupled with available cell type specific promoters, makes TRAP a useful approach to enable the discovery of molecular components in response to external or genetic perturbations.


Assuntos
Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/isolamento & purificação , Perfilação da Expressão Gênica/métodos , Ribossomos/genética , Transcriptoma/fisiologia , Animais , Caenorhabditis elegans/citologia , Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Ribossomos/metabolismo
10.
Nat Methods ; 10(8): 741-3, 2013 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-23817069

RESUMO

We report the use of clustered, regularly interspaced, short palindromic repeats (CRISPR)-associated endonuclease Cas9 to target genomic sequences in the Caenorhabditis elegans germ line using single-guide RNAs that are expressed from a U6 small nuclear RNA promoter. Our results demonstrate that targeted, heritable genetic alterations can be achieved in C. elegans, providing a convenient and effective approach for generating loss-of-function mutants.


Assuntos
Caenorhabditis elegans/genética , Genoma , Sequências Repetidas Invertidas , RNA Nuclear Pequeno/genética , Animais , Engenharia Genética/métodos , Regiões Promotoras Genéticas , Proteínas de Peixe-Zebra/genética , Pequeno RNA não Traduzido
11.
Nature ; 465(7294): 53-9, 2010 May 06.
Artigo em Inglês | MEDLINE | ID: mdl-20445623

RESUMO

Alternative splicing has a crucial role in the generation of biological complexity, and its misregulation is often involved in human disease. Here we describe the assembly of a 'splicing code', which uses combinations of hundreds of RNA features to predict tissue-dependent changes in alternative splicing for thousands of exons. The code determines new classes of splicing patterns, identifies distinct regulatory programs in different tissues, and identifies mutation-verified regulatory sequences. Widespread regulatory strategies are revealed, including the use of unexpectedly large combinations of features, the establishment of low exon inclusion levels that are overcome by features in specific tissues, the appearance of features deeper into introns than previously appreciated, and the modulation of splice variant levels by transcript structure characteristics. The code detected a class of exons whose inclusion silences expression in adult tissues by activating nonsense-mediated messenger RNA decay, but whose exclusion promotes expression during embryogenesis. The code facilitates the discovery and detailed characterization of regulated alternative splicing events on a genome-wide scale.


Assuntos
Processamento Alternativo/genética , Regulação da Expressão Gênica , Código Genético/genética , Modelos Genéticos , RNA Mensageiro/metabolismo , Animais , Inativação Gênica , Humanos , Camundongos , Reprodutibilidade dos Testes
12.
Adv Exp Med Biol ; 907: 229-61, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27256389

RESUMO

Alternative pre-mRNA splicing serves as an elegant mechanism for generating transcriptomic and proteomic diversity between cell and tissue types. In this chapter, we highlight key concepts and outstanding goals in studies of tissue and cell-specific alternative splicing. We place particular emphasis on the use of C. elegans as a tractable model organism for in vivo studies of alternative splicing between tissues and also at single cell resolution. We describe our current understanding of tissue and cell-specific regulation in the animal, and emerging techniques that will allow for future mechanistic studies as well as systems level investigations of spatio-temporal splicing under laboratory conditions and in response to environmental stimuli.


Assuntos
Processamento Alternativo , Proteínas de Caenorhabditis elegans/fisiologia , Caenorhabditis elegans/genética , Regulação da Expressão Gênica/genética , Fatores de Processamento de RNA/fisiologia , RNA de Helmintos/metabolismo , RNA Mensageiro/metabolismo , Animais , Evolução Biológica , Éxons , Genes Reporter , Humanos , Invertebrados/genética , Especificidade de Órgãos , RNA/genética , RNA/metabolismo , Precursores de RNA/genética , Precursores de RNA/metabolismo , RNA de Helmintos/genética , RNA Mensageiro/genética , Proteínas de Ligação a RNA/fisiologia , Especificidade da Espécie , Spliceossomos/metabolismo , Vertebrados/genética
13.
J Neurosci ; 34(40): 13364-70, 2014 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-25274815

RESUMO

Learning is an essential function of the nervous system. However, our understanding of molecular underpinnings of learning remains incomplete. Here, we characterize a conserved protein EOL-1 that regulates olfactory learning in Caenorhabditis elegans. A recessive allele of eol-1 (enhanced olfactory learning) learns better to adjust its olfactory preference for bacteria foods and eol-1 acts in the URX sensory neurons to regulate learning. The mammalian homolog of EOL-1, Dom3Z, which regulates quality control of pre-mRNAs, can substitute the function of EOL-1 in learning regulation, demonstrating functional conservation between these homologs. Mutating the residues of Dom3Z that are critical for its enzymatic activity, and the equivalent residues in EOL-1, abolishes the function of these proteins in learning. Together, our results provide insights into the function of EOL-1/Dom3Z and suggest that its activity in pre-mRNA quality control is involved in neural plasticity.


Assuntos
Aprendizagem da Esquiva/fisiologia , Proteínas de Caenorhabditis elegans/genética , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Condutos Olfatórios/fisiologia , Análise de Variância , Animais , Animais Geneticamente Modificados , Aprendizagem da Esquiva/efeitos dos fármacos , Butanonas/efeitos adversos , Caenorhabditis elegans , Quimiotaxia/efeitos dos fármacos , Quimiotaxia/genética , Exorribonucleases , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Humanos , Camundongos , Mutagênese , Mutação/genética , Condutos Olfatórios/efeitos dos fármacos , Precursores de RNA/metabolismo , Fatores de Tempo
14.
Genome Res ; 21(2): 342-8, 2011 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-21177968

RESUMO

Alternative splicing (AS) plays a crucial role in the diversification of gene function and regulation. Consequently, the systematic identification and characterization of temporally regulated splice variants is of critical importance to understanding animal development. We have used high-throughput RNA sequencing and microarray profiling to analyze AS in C. elegans across various stages of development. This analysis identified thousands of novel splicing events, including hundreds of developmentally regulated AS events. To make these data easily accessible and informative, we constructed the C. elegans Splice Browser, a web resource in which researchers can mine AS events of interest and retrieve information about their relative levels and regulation across development. The data presented in this study, along with the Splice Browser, provide the most comprehensive set of annotated splice variants in C. elegans to date, and are therefore expected to facilitate focused, high resolution in vivo functional assays of AS function.


Assuntos
Processamento Alternativo/genética , Caenorhabditis elegans/genética , Animais , Bases de Dados Genéticas , Éxons/genética , Feminino , Perfilação da Expressão Gênica , Estudo de Associação Genômica Ampla , Masculino , Dados de Sequência Molecular , Análise de Sequência com Séries de Oligonucleotídeos , Software
15.
RNA ; 17(5): 775-91, 2011 May.
Artigo em Inglês | MEDLINE | ID: mdl-21415141

RESUMO

Recent genome-wide analyses have indicated that almost all primary transcripts from multi-exon human genes undergo alternative pre-mRNA splicing (AS). Given the prevalence of AS and its importance in expanding proteomic complexity, a major challenge that lies ahead is to determine the functional specificity of isoforms in a cellular context. A significant fraction of alternatively spliced transcripts are regulated in a tissue- or cell-type-specific manner, suggesting that these mRNA variants likely function in the generation of cellular diversity. Complementary to these observations, several tissue-specific splicing factors have been identified, and a number of methodological advances have enabled the identification of large repertoires of target transcripts regulated by these proteins. An emerging theme is that tissue-specific splicing factors regulate coherent sets of splice variants in genes known to function in related biological pathways. This review focuses on the recent progress in our understanding of neural-specific splicing factors and their regulatory networks and outlines existing and emerging strategies for uncovering important biological roles for the isoforms that comprise these networks.


Assuntos
Processamento Alternativo , Redes Reguladoras de Genes , Neurônios , Transcrição Gênica , Animais , Genoma , Humanos , Neurônios/metabolismo , Especificidade de Órgãos
16.
bioRxiv ; 2023 Jan 22.
Artigo em Inglês | MEDLINE | ID: mdl-36711719

RESUMO

Neurons modify their transcriptomes in response to an animal’s experience. How specific experiences are transduced to modulate gene expression and precisely tune neuronal functions are not fully defined. Here, we describe the molecular profile of a thermosensory neuron pair in C. elegans experiencing different temperature stimuli. We find that distinct salient features of the temperature stimulus including its duration, magnitude of change, and absolute value are encoded in the gene expression program in this single neuron, and identify a novel transmembrane protein and a transcription factor whose specific transcriptional dynamics are essential to drive neuronal, behavioral, and developmental plasticity. Expression changes are driven by broadly expressed activity-dependent transcription factors and corresponding cis -regulatory elements that nevertheless direct neuron- and stimulus-specific gene expression programs. Our results indicate that coupling of defined stimulus characteristics to the gene regulatory logic in individual specialized neuron types can customize neuronal properties to drive precise behavioral adaptation.

17.
Microsyst Nanoeng ; 9: 17, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-36844938

RESUMO

Caenorhabditis elegans embryos have been widely used to study cellular processes and developmental regulation at early stages. However, most existing microfluidic devices focus on the studies of larval or adult worms rather than embryos. To accurately study the real-time dynamics of embryonic development under different conditions, many technical barriers must be overcome; these can include single-embryo sorting and immobilization, precise control of the experimental environment, and long-term live imaging of embryos. This paper reports a spiral microfluidic device for effective sorting, trapping, and long-term live imaging of single C. elegans embryos under precisely controlled experimental conditions. The device successfully sorts embryos from a mixed population of C. elegans at different developmental stages via Dean vortices generated inside a spiral microchannel and traps the sorted embryos at single-cell resolution through hydrodynamic traps on the sidewall of the spiral channel for long-term imaging. Through the well-controlled microenvironment inside the microfluidic device, the response of the trapped C. elegans embryos to mechanical and chemical stimulation can be quantitatively measured. The experimental results show that a gentle hydrodynamic force would induce faster growth of embryos, and embryos developmentally arrested in the high-salinity solution could be rescued by the M9 buffer. The microfluidic device provides new avenues for easy, rapid, high-content screening of C. elegans embryos.

18.
Curr Biol ; 33(8): 1487-1501.e7, 2023 04 24.
Artigo em Inglês | MEDLINE | ID: mdl-36977417

RESUMO

Neurons modify their transcriptomes in response to an animal's experience. How specific experiences are transduced to modulate gene expression and precisely tune neuronal functions are not fully defined. Here, we describe the molecular profile of a thermosensory neuron pair in C. elegans experiencing different temperature stimuli. We find that distinct salient features of the temperature stimulus, including its duration, magnitude of change, and absolute value, are encoded in the gene expression program in this single neuron type, and we identify a novel transmembrane protein and a transcription factor whose specific transcriptional dynamics are essential to drive neuronal, behavioral, and developmental plasticity. Expression changes are driven by broadly expressed activity-dependent transcription factors and corresponding cis-regulatory elements that nevertheless direct neuron- and stimulus-specific gene expression programs. Our results indicate that coupling of defined stimulus characteristics to the gene regulatory logic in individual specialized neuron types can customize neuronal properties to drive precise behavioral adaptation.


Assuntos
Proteínas de Caenorhabditis elegans , Caenorhabditis elegans , Animais , Caenorhabditis elegans/fisiologia , Proteínas de Caenorhabditis elegans/metabolismo , Células Receptoras Sensoriais/fisiologia , Temperatura
20.
Methods Mol Biol ; 2537: 211-229, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35895267

RESUMO

Alternative splicing is a key layer of gene regulation that is frequently modulated in a spatiotemporal manner. As such, it is a major goal to understand the mechanisms controlling alternative splicing in specific cellular contexts. Reporters that recapitulate alternative splicing patterns of endogenous transcripts have served as excellent tools for dissecting regulatory mechanisms of splicing. In this chapter, we describe a two-color fluorescent reporter system that enables the visualization of alternative splicing patterns by microscopy at single-cell resolution in live animals. We present this reporter system in the context of the model nematode C. elegans.


Assuntos
Processamento Alternativo , Caenorhabditis elegans , Animais , Caenorhabditis elegans/genética , Genes Reporter , Proteínas de Fluorescência Verde/análise , Proteínas de Fluorescência Verde/genética
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