RESUMEN
Coordination of gene regulatory networks is necessary for proper execution of cellular programs throughout development. RNA interference (RNAi) is an essential regulatory mechanism in all metazoans. Proper RNAi-mediated gene regulation requires coordination of several RNAi branches to ensure homeostasis. For example, in Caenorhabditis elegans, the Argonautes, ALG-3 and ALG-4, are expressed specifically during spermatogenesis (L4 stage) and bind small interfering RNAs (siRNAs) complementary to sperm-enriched genes. We find that alg-3 and alg-4 are regulated by siRNAs. Our work shows that gene switches are operated via these siRNAs to regulate the Argonautes' expression in a temporal manner. This RNAi-to-RNAi regulatory cascade is essential for coordinating ALG-3/4 pathway function, particularly during heat stress, to provide thermotolerant sperm-based fertility. This work provides insight into one regulatory motif used to maintain RNAi homeostasis, across developmental stages, despite environmental stressors. As RNAi pathways are evolutionarily conserved, other species likely use similar regulatory architectures to maintain RNAi homeostasis.
RESUMEN
RNA interference (RNAi) is an essential regulatory mechanism in all animals. In Caenorhabditis elegans, several classes of small RNAs act to silence or license expression of mRNA targets. ERI-6/7 is required for the production of some endogenous small interfering RNAs (siRNAs) and acts as a negative regulator of the exogenous RNAi pathway. We find that the genomic locus encoding eri-6/7 contains two distinct regions that are targeted by endogenous siRNAs. Loss of these siRNAs disrupts eri-6/7 mRNA expression, resulting in increased production of siRNAs from other small RNA pathways because these pathways compete with eri-6/7-dependent transcripts for access to the downstream siRNA amplification machinery. Thus, the pathway acts like a small-RNA-mediated feedback loop to ensure homeostasis of gene expression by small RNA pathways. Similar feedback loops that maintain chromatin homeostasis have been identified in yeast and Drosophila melanogaster, suggesting an evolutionary conservation of feedback mechanisms in gene regulatory pathways.
Asunto(s)
Proteínas de Caenorhabditis elegans/metabolismo , ADN Helicasas/metabolismo , Interferencia de ARN/fisiología , Animales , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/genética , ADN Helicasas/genética , Retroalimentación , Silenciador del Gen/fisiología , ARN Bicatenario/genética , ARN Bicatenario/metabolismo , ARN Mensajero/metabolismo , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismoRESUMEN
piRNAs play a critical role in the regulation of transposons and other germline genes. In Caenorhabditis elegans, regulation of piRNA target genes is mediated by the mutator complex, which synthesizes high levels of siRNAs through the activity of an RNA-dependent RNA polymerase. However, the steps between mRNA recognition by the piRNA pathway and siRNA amplification by the mutator complex are unknown. Here, we identify the Tudor domain protein, SIMR-1, as acting downstream of piRNA production and upstream of mutator complex-dependent siRNA biogenesis. Interestingly, SIMR-1 also localizes to distinct subcellular foci adjacent to P granules and Mutator foci, two phase-separated condensates that are the sites of piRNA-dependent mRNA recognition and mutator complex-dependent siRNA amplification, respectively. Thus, our data suggests a role for multiple perinuclear condensates in organizing the piRNA pathway and promoting mRNA regulation by the mutator complex.
In the biological world, a process known as RNA interference helps cells to switch genes on and off and to defend themselves against harmful genetic material. This mechanism works by deactivating RNA sequences, the molecular templates cells can use to create proteins. Overall, RNA interference relies on the cell creating small RNA molecules that can target and inhibit the harmful RNA sequences that need to be silenced. More precisely, in round worms such as Caenorhabditis elegans, RNA interference happens in two steps. First, primary small RNAs identify the target sequences, which are then combatted by newly synthetised, secondary small RNAs. A number of proteins are also involved in both steps of the process. RNA interference is particularly important to preserve fertility, guarding sex cells against 'rogue' segments of genetic information that could be passed on to the next generation. In future sex cells, the proteins involved in RNA interference cluster together, forming a structure called a germ granule. Yet, little is known about the roles and identity of these proteins. To fill this knowledge gap, Manage et al. focused on the second stage of the RNA interference pathway in the germ granules of C. elegans, examining the molecules that physically interact with a key protein. This work revealed a new protein called SIMR-1. Looking into the role of SIMR-1 showed that the protein is required to amplify secondary small RNAs, but not to identify target sequences. However, it only promotes the creation of secondary small RNAs if a specific subtype of primary small RNAs have recognized the target RNAs for silencing. Further experiments also showed that within the germ granule, SIMR-1 is present in a separate substructure different from any compartment previously identified. This suggests that each substep of the RNA interference process takes place at a different location in the granule. In both C. elegans and humans, disruptions in the RNA interference pathway can lead to conditions such as cancer or infertility. Dissecting the roles of the proteins involved in this process in roundworms may help to better grasp how this process unfolds in mammals, and how it could be corrected in the case of disease.
Asunto(s)
Proteínas de Caenorhabditis elegans/genética , Caenorhabditis elegans/genética , ARN Mensajero/genética , ARN Interferente Pequeño/genética , Dominio Tudor/genética , Animales , Femenino , MasculinoRESUMEN
Repression of cellular reprogramming in germ cells is critical to maintaining cell fate and fertility. When germ cells mis-express somatic genes they can be directly converted into other cell types, resulting in loss of totipotency and reproductive potential. Identifying the molecular mechanisms that coordinate these cell fate decisions is an active area of investigation. Here we show that RNAi pathways play a key role in maintaining germline gene expression and totipotency after heat stress. By examining transcriptional changes that occur in mut-16 mutants, lacking a key protein in the RNAi pathway, at elevated temperature we found that genes normally expressed in the soma are mis-expressed in germ cells. Furthermore, these genes displayed increased chromatin accessibility in the germlines of mut-16 mutants at elevated temperature. These findings indicate that the RNAi pathway plays a key role in preventing aberrant expression of somatic genes in the germline during heat stress. This regulation occurs in part through the maintenance of germline chromatin, likely acting through the nuclear RNAi pathway. Identification of new pathways governing germ cell reprogramming is critical to understanding how cells maintain proper gene expression and may provide key insights into how cell identity is lost in some germ cell tumors.
Asunto(s)
Caenorhabditis elegans/genética , Células Germinativas/metabolismo , Respuesta al Choque Térmico/genética , Interferencia de ARN , Animales , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/genética , Cromatina/metabolismo , Regulación de la Expresión Génica , Secuencias Repetitivas Esparcidas , Mutación , Oogénesis/genética , ARN Mensajero/metabolismo , ARN Pequeño no Traducido/metabolismo , Espermatogénesis/genética , Regulación hacia ArribaRESUMEN
Regulation of transcription is the main mechanism responsible for precise control of gene expression. Whereas the majority of transcriptional regulation is mediated by DNA-binding transcription factors that bind to regulatory gene regions, an elegant alternative strategy employs small RNA guides, Piwi-interacting RNAs (piRNAs) to identify targets of transcriptional repression. Here, we show that in Drosophila the small ubiquitin-like protein SUMO and the SUMO E3 ligase Su(var)2-10 are required for piRNA-guided deposition of repressive chromatin marks and transcriptional silencing of piRNA targets. Su(var)2-10 links the piRNA-guided target recognition complex to the silencing effector by binding the piRNA/Piwi complex and inducing SUMO-dependent recruitment of the SetDB1/Wde histone methyltransferase effector. We propose that in Drosophila, the nuclear piRNA pathway has co-opted a conserved mechanism of SUMO-dependent recruitment of the SetDB1/Wde chromatin modifier to confer repression of genomic parasites.
Asunto(s)
Proteínas de Drosophila/metabolismo , Proteínas Inhibidoras de STAT Activados/metabolismo , ARN Interferente Pequeño/genética , Proteínas Modificadoras Pequeñas Relacionadas con Ubiquitina/metabolismo , Animales , Proteínas Argonautas/metabolismo , Núcleo Celular/metabolismo , Cromatina/genética , Cromatina/metabolismo , Elementos Transponibles de ADN , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Regulación de la Expresión Génica/genética , Silenciador del Gen/fisiología , Unión Proteica , Proteínas Inhibidoras de STAT Activados/genética , ARN Interferente Pequeño/metabolismo , Proteínas Modificadoras Pequeñas Relacionadas con Ubiquitina/genética , Transcripción Genética/genéticaRESUMEN
Animals, including humans, can adapt to environmental stress through phenotypic plasticity. The free-living nematode Caenorhabditis elegans can adapt to harsh environments by undergoing a whole-animal change, involving exiting reproductive development and entering the stress-resistant dauer larval stage. The dauer is a dispersal stage with dauer-specific behaviors for finding and stowing onto carrier animals, but how dauers acquire these behaviors, despite having a physically limited nervous system of 302 neurons, is poorly understood. We compared dauer and reproductive development using whole-animal RNA sequencing at fine time points and at sufficient depth to measure transcriptional changes within single cells. We detected 8,042 genes differentially expressed during dauer and reproductive development and observed striking up-regulation of neuropeptide genes during dauer entry. We knocked down neuropeptide processing using sbt-1 mutants and demonstrate that neuropeptide signaling promotes the decision to enter dauer rather than reproductive development. We also demonstrate that during dauer neuropeptides modulate the dauer-specific nictation behavior (carrier animal-hitchhiking) and are necessary for switching from repulsion to CO2 (a carrier animal cue) in nondauers to CO2 attraction in dauers. We tested individual neuropeptides using CRISPR knockouts and existing strains and demonstrate that the combined effects of flp-10 and flp-17 mimic the effects of sbt-1 on nictation and CO2 attraction. Through meta-analysis, we discovered similar up-regulation of neuropeptides in the dauer-like infective juveniles of diverse parasitic nematodes, suggesting the antiparasitic target potential of SBT-1. Our findings reveal that, under stress, increased neuropeptide signaling in C. elegans enhances their decision-making accuracy and expands their behavioral repertoire.
Asunto(s)
Caenorhabditis elegans/fisiología , Neuropéptidos/fisiología , Animales , Caenorhabditis elegans/genética , Caenorhabditis elegans/crecimiento & desarrollo , Dióxido de Carbono/metabolismo , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas , FMRFamida/química , Técnicas de Silenciamiento del Gen , Genes de Helminto , Larva/crecimiento & desarrollo , Larva/fisiología , Neuropéptidos/genética , Fenotipo , ARN de Helminto , Reproducción , Análisis de Secuencia de ARN , Transducción de Señal , Estrés FisiológicoRESUMEN
The piRNA pathway represses transposable elements in the gonads and thereby plays a vital role in protecting the integrity of germline genomes of animals. Mature piRNAs are processed from longer transcripts, piRNA precursors (pre-piRNAs). In Drosophila, processing of pre-piRNAs is initiated by piRNA-guided Slicer cleavage or the endonuclease Zucchini (Zuc). As Zuc does not have any sequence or structure preferences in vitro, it is not known how piRNA precursors are selected and channeled into the Zuc-dependent processing pathway. We show that a heterologous RNA that lacks complementary piRNAs is processed into piRNAs upon recruitment of several piRNA pathway factors. This processing requires Zuc and the helicase Armitage (Armi). Aubergine (Aub), Argonaute 3 (Ago3), and components of the nuclear RDC complex, which are required for normal piRNA biogenesis in germ cells, are dispensable. Our approach allows discrimination of proteins involved in the transcription and export of piRNA precursors from components required for the cytoplasmic processing steps. piRNA processing correlates with localization of the substrate RNA to nuage, a distinct membraneless cytoplasmic compartment, which surrounds the nucleus of germ cells, suggesting that sequestration of RNA to this subcellular compartment is both necessary and sufficient for selecting piRNA biogenesis substrates.
Asunto(s)
Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Endorribonucleasas/metabolismo , ARN Helicasas/metabolismo , Precursores del ARN/metabolismo , Procesamiento Postranscripcional del ARN , ARN Interferente Pequeño/biosíntesis , Animales , Proteínas Argonautas/genética , Proteínas Argonautas/metabolismo , Citoplasma/metabolismo , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Endorribonucleasas/genética , Femenino , Células Germinativas/metabolismo , Ovario/metabolismo , Factores de Iniciación de Péptidos/genética , Factores de Iniciación de Péptidos/metabolismo , ARN Helicasas/genéticaRESUMEN
In the metazoan germline, piwi proteins and associated piwi-interacting RNAs (piRNAs) provide a defense system against the expression of transposable elements. In the cytoplasm, piRNA sequences guide piwi complexes to destroy complementary transposon transcripts by endonucleolytic cleavage. However, some piwi family members are nuclear, raising the possibility of alternative pathways for piRNA-mediated regulation of gene expression. We found that Drosophila Piwi is recruited to chromatin, colocalizing with RNA polymerase II (Pol II) on polytene chromosomes. Knockdown of Piwi in the germline increases expression of transposable elements that are targeted by piRNAs, whereas protein-coding genes remain largely unaffected. Derepression of transposons upon Piwi depletion correlates with increased occupancy of Pol II on their promoters. Expression of piRNAs that target a reporter construct results in a decrease in Pol II occupancy and an increase in repressive H3K9me3 marks and heterochromatin protein 1 (HP1) on the reporter locus. Our results indicate that Piwi identifies targets complementary to the associated piRNA and induces transcriptional repression by establishing a repressive chromatin state when correct targets are found.
Asunto(s)
Proteínas Argonautas/metabolismo , Cromatina/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Silenciador del Gen , ARN Interferente Pequeño/metabolismo , Animales , Núcleo Celular/genética , Núcleo Celular/metabolismo , Femenino , Cromosomas Politénicos/metabolismo , ARN Interferente Pequeño/genéticaRESUMEN
BACKGROUND: Cell polarity genes including Crumbs (Crb) and Par complexes are essential for controlling photoreceptor morphogenesis. Among the Crb and Par complexes, Bazooka (Baz, Par-3 homolog) acts as a nodal component for other cell polarity proteins. Therefore, finding other genes interacting with Baz will help us to understand the cell polarity genes' role in photoreceptor morphogenesis. METHODOLOGY/PRINCIPAL FINDINGS: Here, we have found a genetic interaction between baz and centrosomin (cnn). Cnn is a core protein for centrosome which is a major microtubule-organizing center. We analyzed the effect of the cnn mutation on developing eyes to determine its role in photoreceptor morphogenesis. We found that Cnn is dispensable for retinal differentiation in eye imaginal discs during the larval stage. However, photoreceptors deficient in Cnn display dramatic morphogenesis defects including the mislocalization of Crumbs (Crb) and Bazooka (Baz) during mid-stage pupal eye development, suggesting that Cnn is specifically required for photoreceptor morphogenesis during pupal eye development. This role of Cnn in apical domain modulation was further supported by Cnn's gain-of-function phenotype. Cnn overexpression in photoreceptors caused the expansion of the apical Crb membrane domain, Baz and adherens junctions (AJs). CONCLUSIONS/SIGNIFICANCE: These results strongly suggest that the interaction of Baz and Cnn is essential for apical domain and AJ modulation during photoreceptor morphogenesis, but not for the initial photoreceptor differentiation in the Drosophila photoreceptor.