RESUMEN
Movement is a key feature of animal systems, yet its embryonic origins are not fully understood. Here, we investigate the genetic basis underlying the embryonic onset of movement in Drosophila focusing on the role played by small non-coding RNAs (microRNAs, miRNAs). To this end, we first develop a quantitative behavioural pipeline capable of tracking embryonic movement in large populations of fly embryos, and using this system, discover that the Drosophila miRNA miR-2b-1 plays a role in the emergence of movement. Through the combination of spectral analysis of embryonic motor patterns, cell sorting and RNA in situs, genetic reconstitution tests, and neural optical imaging we define that miR-2b-1 influences the emergence of embryonic movement by exerting actions in the developing nervous system. Furthermore, through the combination of bioinformatics coupled to genetic manipulation of miRNA expression and phenocopy tests we identify a previously uncharacterised (but evolutionarily conserved) chloride channel encoding gene - which we term Movement Modulator (Motor) - as a genetic target that mechanistically links miR-2b-1 to the onset of movement. Cell-specific genetic reconstitution of miR-2b-1 expression in a null miRNA mutant background, followed by behavioural assays and target gene analyses, suggest that miR-2b-1 affects the emergence of movement through effects in sensory elements of the embryonic circuitry, rather than in the motor domain. Our work thus reports the first miRNA system capable of regulating embryonic movement, suggesting that other miRNAs are likely to play a role in this key developmental process in Drosophila as well as in other species.
Asunto(s)
MicroARNs , Animales , MicroARNs/metabolismo , MicroARNs/genética , Drosophila melanogaster/genética , Drosophila melanogaster/embriología , Regulación del Desarrollo de la Expresión Génica , Movimiento , Embrión no Mamífero/metabolismo , Drosophila/genética , Drosophila/embriología , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismoRESUMEN
The molecular mechanisms underlying the stability of mature neurons and neural circuits are poorly understood. Here we explore this problem and discover that the Hox genes are a component of the genetic program that maintains normal neural function in adult Drosophila. We show that post-developmental downregulation of the Hox gene Ultrabithorax (Ubx) in adult neurons leads to substantial anomalies in flight. Mapping the cellular basis of these effects reveals that Ubx is required within a subset of dopaminergic neurons, and cell circuitry analyses in combination with optogenetics allow us to link these dopaminergic neurons to flight control. Functional imaging experiments show that Ubx is necessary for normal dopaminergic activity, and neuron-specific RNA-sequencing defines two previously uncharacterized ion channel-encoding genes as potential mediators of Ubx behavioral roles. Our study thus reveals a novel role of the Hox system in controlling adult behavior and neural function. Based on the broad evolutionary conservation of the Hox system across distantly related animal phyla, we predict that the Hox genes might play neurophysiological roles in adult forms of other species, including humans.
Asunto(s)
Proteínas de Drosophila , Genes Homeobox , Animales , Evolución Biológica , Neuronas Dopaminérgicas , Drosophila/genética , Proteínas de Drosophila/genética , Genes Homeobox/genética , Proteínas de Homeodominio/genética , Optogenética , Factores de Transcripción/genéticaRESUMEN
All what we see, touch, hear, taste, or smell must first be detected by the sensory elements of our nervous system. Sensory neurons, therefore, represent a critical component in all neural circuits and their correct function is essential for the generation of behavior and adaptation to the environment. Here, we report that the evolutionarily-conserved microRNA (miRNA) miR-263b plays a key behavioral role in Drosophila melanogaster through effects on the function of larval sensory neurons. Several independent experiments (in 50:50 male:female populations) support this finding: first, miRNA expression analysis, via reporter expression and fluorescent-activated cell sorting (FACS)-quantitative PCR (qPCR) analysis, demonstrate miR-263b expression in larval sensory neurons. Second, behavioral tests in miR-263b null mutants show defects in self-righting, an innate and evolutionarily conserved posture-control behavior that allows larvae to rectify their position if turned upside-down. Third, competitive inhibition of miR-263b in sensory neurons using a miR-263b "sponge" leads to self-righting defects. Fourth, systematic analysis of sensory neurons in miR-263b mutants shows no detectable morphologic defects in their stereotypic pattern, while genetically-encoded calcium sensors expressed in the sensory domain reveal a reduction in neural activity in miR-263b mutants. Fifth, miR-263b null mutants show reduced "touch-response" behavior and a compromised response to sound, both characteristic of larval sensory deficits. Furthermore, bioinformatic miRNA target analysis, gene expression assays, and behavioral phenocopy experiments suggest that miR-263b might exert its effects, at least in part, through repression of the basic helix-loop-helix (bHLH) transcription factor Atonal Altogether, our study suggests a model in which miRNA-dependent control of transcription factor expression affects sensory function and behavior.SIGNIFICANCE STATEMENT Sensory neurons are key to neural circuit function, but how these neurons acquire their specific properties is not well understood. Here, we examine this problem, focusing on the roles played by microRNAs (miRNAs). Using Drosophila, we demonstrate that the evolutionarily-conserved miRNA miR-263b controls sensory neuron function allowing the animal to perform an adaptive, elaborate three-dimensional movement. Our work thus shows that microRNAs can control complex motor behaviors by modulating sensory neuron physiology, and suggests that similar miRNA-dependent mechanisms may operate in other species. The work contributes to advance the understanding of the molecular basis of behavior and the biological roles of microRNAs within the nervous system.
Asunto(s)
MicroARNs/biosíntesis , Movimiento/fisiología , Postura/fisiología , Reflejo de Enderezamiento/fisiología , Células Receptoras Sensoriales/fisiología , Animales , Drosophila melanogaster , Femenino , Masculino , MicroARNs/genéticaRESUMEN
Movement is the main output of the nervous system. It emerges during development to become a highly coordinated physiological process essential to survival and adaptation of the organism to the environment. Similar movements can be observed in morphologically distinct developmental stages of an organism, but it is currently unclear whether or not these movements have a common molecular cellular basis. Here we explore this problem in Drosophila, focusing on the roles played by the microRNA (miRNA) locus miR-iab4/8, which we previously showed to be essential for the normal corrective response displayed by the fruit fly larva when turned upside down (self-righting). Our study shows that miR-iab4 is required for normal self-righting across all three Drosophila larval stages. Unexpectedly, we also discover that this miRNA is essential for normal self-righting behavior in the adult fly, an organism with different morphology, neural constitution, and biomechanics. Through the combination of gene expression, optical imaging, and quantitative behavioral approaches, we provide evidence that miR-iab4 exerts its effects on adult self-righting behavior in part through repression of the Hox gene Ultrabithorax (Ubx) in a specific set of adult motor neurons, the NB2-3/lin15 neurons. Our results show that miRNA controls the function, rather than the morphology, of these neurons and demonstrate that post-developmental changes in Hox gene expression can modulate behavior in the adult. Our work reveals that a common miRNA-Hox genetic module can be re-deployed in different neurons to control functionally equivalent movements in biomechanically distinct organisms and describes a novel post-developmental role of the Hox genes in adult neural function.
Asunto(s)
Proteínas de Drosophila/genética , Drosophila melanogaster/fisiología , Proteínas de Homeodominio/genética , MicroARNs/genética , Factores de Transcripción/genética , Animales , Fenómenos Biomecánicos , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/genética , Drosophila melanogaster/crecimiento & desarrollo , Redes Reguladoras de Genes/genética , Genes Homeobox/fisiología , Proteínas de Homeodominio/metabolismo , Larva/fisiología , MicroARNs/metabolismo , Movimiento/fisiología , Factores de Transcripción/metabolismoRESUMEN
Disruptions of normal Hox gene expression can lead to severe morphological defects, revealing a link between the regulation of Hox expression and pattern formation. Here, we explore these links, focusing on the impact of microRNA regulation on the expression of the Drosophila Hox gene Ultrabithorax (Ubx) during haltere development. Through a combination of bioinformatic and transcriptomic analyses, we identify the miR-310/313 cluster (miR-310C) as a candidate regulator of Ubx Several experiments confirm this. First, miR-310C and Ubx protein show complementary expression patterns in haltere imaginal discs; second, artificial activation of miR-310C expression in haltere discs leads to Ubx-like phenotypes. Third, expression of a fluorescent reporter bearing Ubx 3'UTR sequences is reduced when co-expressed with miR-310C Fourth, deletion of miR-310C leads to Ubx upregulation and changes the array of mechanosensory sensilla at the base of the haltere. Fifth, an artificial increase of Ubx levels within the miR-310C expression domain phenocopies the mechanosensory defects observed in miR-310C mutants. We propose that miR-310C-mediated repression delimits Ubx fine-grain expression, contributing to the sculpting of complex morphologies in the Drosophila haltere with implications for flight control. Our work reveals a novel role of microRNA regulation in the control of Hox gene expression with impact on morphology.
Asunto(s)
Proteínas de Drosophila/genética , Drosophila melanogaster/anatomía & histología , Drosophila melanogaster/genética , Regulación del Desarrollo de la Expresión Génica , Genes Homeobox , Proteínas de Homeodominio/genética , MicroARNs/metabolismo , Factores de Transcripción/genética , Alas de Animales/anatomía & histología , Regiones no Traducidas 3'/genética , Animales , Tipificación del Cuerpo/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/ultraestructura , Sitios Genéticos , Proteínas de Homeodominio/metabolismo , MicroARNs/genética , Mutación/genética , Fenotipo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Factores de Transcripción/metabolismo , Regulación hacia Arriba/genética , Alas de Animales/ultraestructuraRESUMEN
The effects of microRNA (miRNA) regulation on the genetic programs underlying behavior remain largely unexplored. Despite this, recent work in Drosophila shows that mutation of a single miRNA locus (miR-iab4/iab8) affects the capacity of the larva to correct its orientation if turned upside down (self-righting, SR), suggesting that other miRNAs might also be involved in behavioral control. Here we explore this possibility, studying early larval SR behavior in a collection of 81 Drosophila miRNA mutants covering almost the entire miRNA complement of the late embryo. Unexpectedly, we observe that >40% of all miRNAs tested significantly affect SR time, revealing pervasive behavioral effects of miRNA regulation in the early larva. Detailed analyses of those miRNAs affecting SR behavior (SR-miRNAs) show that individual miRNAs can affect movement in different ways, suggesting that specific molecular and cellular elements are affected by individual miRNA mutations. Furthermore, gene expression analysis shows that the Hox gene Abdominal-B (Abd-B) represents one of the targets deregulated by several SR-miRNAs. Our work thus reveals pervasive effects of miRNA regulation on a complex innate behavior in Drosophila and suggests that miRNAs may be core components of the genetic programs underlying behavioral control in other animals too.
Asunto(s)
Conducta Animal , MicroARNs/genética , Movimiento , Animales , Drosophila/genética , Drosophila/crecimiento & desarrollo , Drosophila/fisiología , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/metabolismo , Larva/metabolismo , Larva/fisiología , MutaciónRESUMEN
Alternative polyadenylation (APA) is a widespread gene regulatory mechanism that generates mRNAs with different 3'-ends, allowing them to interact with different sets of RNA regulators such as microRNAs and RNA-binding proteins. Recent studies have shown that during development, neural tissues produce mRNAs with particularly long 3'UTRs, suggesting that such extensions might be important for neural development and function. Despite this, the mechanisms underlying neural APA are not well understood. Here, we investigate this problem within the Drosophila nervous system, focusing on the roles played by general cleavage and polyadenylation factors (CPA factors). In particular, we examine the model that modulations in CPA factor concentration may affect APA during development. For this, we first analyse the expression of the Drosophila orthologues of all mammalian CPA factors and note that their expression decreases during embryogenesis. In contrast to this global developmental decrease in CPA factor expression, we see that cleavage factor I (CFI) expression is actually elevated in the late embryonic central nervous system, suggesting that CFI might play a special role in neural tissues. To test this, we use the UAS/Gal4 system to deplete CFI proteins from neural tissue and observe that in this condition, multiple genes switch their APA patterns, demonstrating a role of CFI in APA control during Drosophila neural development. Furthermore, analysis of genes with 3'UTR extensions of different length leads us to suggest a novel relation between 3'UTR length and sensitivity to CPA factor expression. Our work thus contributes to the understanding of the mechanisms of APA control within the developing central nervous system.
Asunto(s)
Regiones no Traducidas 3'/genética , Drosophila melanogaster/genética , Regulación del Desarrollo de la Expresión Génica , Sistema Nervioso/metabolismo , Poliadenilación/genética , ARN Mensajero/genética , Factores de Escisión y Poliadenilación de ARNm/genética , Animales , Sistema Nervioso/citología , Factores de Escisión y Poliadenilación de ARNm/metabolismoRESUMEN
Ultraconserved elements (UCEs) are discrete genomic elements conserved across large evolutionary distances. Although UCEs have been linked to multiple facets of mammalian gene regulation their extreme evolutionary conservation remains largely unexplained. Here, we apply a computational approach to investigate this question in Drosophila, exploring the molecular functions of more than 1,500 UCEs shared across the genomes of 12 Drosophila species. Our data indicate that Drosophila UCEs are hubs for gene regulatory functions and suggest that UCE sequence invariance originates from their combinatorial roles in gene control. We also note that the gene regulatory roles of intronic and intergenic UCEs (iUCEs) are distinct from those found in exonic UCEs (eUCEs). In iUCEs, transcription factor (TF) and epigenetic factor binding data strongly support iUCE roles in transcriptional and epigenetic regulation. In contrast, analyses of eUCEs indicate that they are two orders of magnitude more likely than the expected to simultaneously include protein-coding sequence, TF-binding sites, splice sites, and RNA editing sites but have reduced roles in transcriptional or epigenetic regulation. Furthermore, we use a Drosophila cell culture system and transgenic Drosophila embryos to validate the notion of UCE combinatorial regulatory roles using an eUCE within the Hox gene Ultrabithorax and show that its protein-coding region also contains alternative splicing regulatory information. Taken together our experiments indicate that UCEs emerge as a result of combinatorial gene regulatory roles and highlight common features in mammalian and insect UCEs implying that similar processes might underlie ultraconservation in diverse animal taxa.
Asunto(s)
Secuencia Conservada , Drosophila/genética , Genes Reguladores , Animales , Animales Modificados Genéticamente , Secuencia de Bases , Evolución Biológica , Biología Computacional/métodos , Bases de Datos de Ácidos Nucleicos , Epigénesis Genética , Femenino , Genómica/métodos , Masculino , Análisis de Secuencia de ADN/métodosRESUMEN
The relationship between microRNA (miRNA) regulation and the specification of behavior is only beginning to be explored. We found that mutation of a single miRNA locus (miR-iab4/iab8) in Drosophila larvae affects the animal's capacity to correct its orientation if turned upside down (self-righting). One of the miRNA targets involved in this behavior is the Hox gene Ultrabithorax, whose derepression in two metameric neurons leads to self-righting defects. In vivo neural activity analysis reveals that these neurons, the self-righting node (SRN), have different activity patterns in wild type and miRNA mutants, whereas thermogenetic manipulation of SRN activity results in changes in self-righting behavior. Our work thus reveals a miRNA-encoded behavior and suggests that other miRNAs might also be involved in behavioral control in Drosophila and other species.
Asunto(s)
Conducta Animal/fisiología , Drosophila melanogaster/fisiología , MicroARNs/fisiología , Orientación/fisiología , Animales , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Regulación de la Expresión Génica , Sitios Genéticos , Proteínas de Homeodominio/genética , Larva/genética , Larva/fisiología , MicroARNs/genética , Mutación , Neuronas/fisiología , Factores de Transcripción/genéticaRESUMEN
Nonsense-mediated mRNA decay (NMD) is a surveillance mechanism that degrades mRNAs harboring premature termination codons (PTCs). We have conducted a genome-wide RNAi screen in Caenorhabditis elegans that resulted in the identification of five novel NMD genes that are conserved throughout evolution. Two of their human homologs, GNL2 (ngp-1) and SEC13 (npp-20), are also required for NMD in human cells. We also show that the C. elegans gene noah-2, which is present in Drosophila melanogaster but absent in humans, is an NMD factor in fruit flies. Altogether, these data identify novel NMD factors that are conserved throughout evolution, highlighting the complexity of the NMD pathway and suggesting that yet uncovered novel factors may act to regulate this process.
Asunto(s)
Caenorhabditis elegans/genética , Proteínas Portadoras/metabolismo , Drosophila melanogaster/genética , Proteínas de Unión al GTP/metabolismo , Degradación de ARNm Mediada por Codón sin Sentido/fisiología , Proteínas Nucleares/metabolismo , Animales , Animales Modificados Genéticamente , Caenorhabditis elegans/crecimiento & desarrollo , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Proteínas Portadoras/genética , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/embriología , Proteínas del Huevo/genética , Proteínas del Huevo/metabolismo , Embrión no Mamífero , Evolución Molecular , Proteínas de Unión al GTP/genética , Técnicas de Silenciamiento del Gen , Células HeLa , Humanos , Proteínas de Microfilamentos/genética , Proteínas de Microfilamentos/metabolismo , Proteínas Nucleares/genética , Interferencia de ARN , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismoRESUMEN
Myriapods (e.g., centipedes and millipedes) display a simple homonomous body plan relative to other arthropods. All members of the class are terrestrial, but they attained terrestriality independently of insects. Myriapoda is the only arthropod class not represented by a sequenced genome. We present an analysis of the genome of the centipede Strigamia maritima. It retains a compact genome that has undergone less gene loss and shuffling than previously sequenced arthropods, and many orthologues of genes conserved from the bilaterian ancestor that have been lost in insects. Our analysis locates many genes in conserved macro-synteny contexts, and many small-scale examples of gene clustering. We describe several examples where S. maritima shows different solutions from insects to similar problems. The insect olfactory receptor gene family is absent from S. maritima, and olfaction in air is likely effected by expansion of other receptor gene families. For some genes S. maritima has evolved paralogues to generate coding sequence diversity, where insects use alternate splicing. This is most striking for the Dscam gene, which in Drosophila generates more than 100,000 alternate splice forms, but in S. maritima is encoded by over 100 paralogues. We see an intriguing linkage between the absence of any known photosensory proteins in a blind organism and the additional absence of canonical circadian clock genes. The phylogenetic position of myriapods allows us to identify where in arthropod phylogeny several particular molecular mechanisms and traits emerged. For example, we conclude that juvenile hormone signalling evolved with the emergence of the exoskeleton in the arthropods and that RR-1 containing cuticle proteins evolved in the lineage leading to Mandibulata. We also identify when various gene expansions and losses occurred. The genome of S. maritima offers us a unique glimpse into the ancestral arthropod genome, while also displaying many adaptations to its specific life history.
Asunto(s)
Artrópodos/genética , Genoma , Sintenía , Animales , Péptidos y Proteínas de Señalización del Ritmo Circadiano/genética , Metilación de ADN , Evolución Molecular , Femenino , Genoma Mitocondrial , Hormonas/genética , Masculino , Familia de Multigenes , Filogenia , Polimorfismo Genético , Proteínas Quinasas/genética , ARN no Traducido/genética , Receptores Odorantes/genética , Selenoproteínas/genética , Cromosomas Sexuales , Factores de Transcripción/genéticaRESUMEN
The regulated head-to-tail expression of Hox genes provides a coordinate system for the activation of specific programmes of cell differentiation according to axial level. Recent work indicates that Hox expression can be regulated via RNA processing but the underlying mechanisms and biological significance of this form of regulation remain poorly understood. Here we explore these issues within the developing Drosophila central nervous system (CNS). We show that the pan-neural RNA-binding protein (RBP) ELAV (Hu antigen) regulates the RNA processing patterns of the Hox gene Ultrabithorax (Ubx) within the embryonic CNS. Using a combination of biochemical, genetic and imaging approaches we demonstrate that ELAV binds to discrete elements within Ubx RNAs and that its genetic removal reduces Ubx protein expression in the CNS leading to the respecification of cellular subroutines under Ubx control, thus defining for the first time a specific cellular role of ELAV within the developing CNS. Artificial provision of ELAV in glial cells (a cell type that lacks ELAV) promotes Ubx expression, suggesting that ELAV-dependent regulation might contribute to cell type-specific Hox expression patterns within the CNS. Finally, we note that expression of abdominal A and Abdominal B is reduced in elav mutant embryos, whereas other Hox genes (Antennapedia) are not affected. Based on these results and the evolutionary conservation of ELAV and Hox genes we propose that the modulation of Hox RNA processing by ELAV serves to adapt the morphogenesis of the CNS to axial level by regulating Hox expression and consequently activating local programmes of neural differentiation.
Asunto(s)
Proteínas de Drosophila/fisiología , Drosophila melanogaster/embriología , Proteínas ELAV/fisiología , Genes Homeobox , Sistema Nervioso/embriología , Procesamiento Postranscripcional del ARN , Animales , Secuencia de Bases , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/genética , Embrión no Mamífero , Regulación del Desarrollo de la Expresión Génica , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/metabolismo , Datos de Secuencia Molecular , Morfogénesis/genética , Sistema Nervioso/metabolismo , Neurogénesis/genética , Filogenia , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
The distribution of sensory bristles on the thorax of Diptera (true flies) provides a useful model for the study of the evolution of spatial patterns. Large bristles called macrochaetes are arranged into species-specific stereotypical patterns determined via spatially discrete expression of the proneural genes achaete-scute (ac-sc). In Drosophila ac-sc expression is regulated by transcriptional activation at sites where bristle precursors develop and by repression outside of these sites. Three genes, extramacrochaetae (emc), hairy (h) and stripe (sr), involved in repression have been documented. Here we demonstrate that in Drosophila, the repressor genes emc and h, like sr, play an essential role in the development of structures forming part of the flight apparatus. In addition we find that, in Calliphora vicina a species diverged from D. melanogaster by about 100 Myr, spatial expression of emc, h and sr is conserved at the location of development of those structures. Based on these findings we argue, first, that the role emc, h and sr in development of the flight apparatus preceded their activities for macrochaete patterning; second, that species-specific variation in activation and repression of ac-sc expression is evolving in parallel to establish a unique distribution of macrochaetes in each species.
Asunto(s)
Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Tipificación del Cuerpo/genética , Dípteros/fisiología , Proteínas de Drosophila/genética , Vuelo Animal , Proteínas de Insectos/genética , Proteínas Represoras/genética , Animales , Secuencia de Bases , Cartilla de ADN , Dípteros/anatomía & histología , Dípteros/genética , Drosophila melanogaster , Hibridación in Situ , Reacción en Cadena de la PolimerasaRESUMEN
Hox genes encode a family of transcriptional regulators that elicit distinct developmental programmes along the head-to-tail axis of animals. The specific regional functions of individual Hox genes largely reflect their restricted expression patterns, the disruption of which can lead to developmental defects and disease. Here, we examine the spectrum of molecular mechanisms controlling Hox gene expression in model vertebrates and invertebrates and find that a diverse range of mechanisms, including nuclear dynamics, RNA processing, microRNA and translational regulation, all concur to control Hox gene outputs. We propose that this complex multi-tiered regulation might contribute to the robustness of Hox expression during development.
Asunto(s)
Proteínas de Drosophila/metabolismo , Genes Homeobox/fisiología , Animales , Cromatina/genética , Cromatina/metabolismo , Drosophila , Proteínas de Drosophila/genética , Regulación del Desarrollo de la Expresión Génica/genética , Regulación del Desarrollo de la Expresión Génica/fisiología , Genes Homeobox/genética , Humanos , Ratones , MicroARNs/genéticaRESUMEN
Current understanding of the molecular mechanisms underlying mRNA degradation indicates that specific mRNA degradation rates are primarily encoded within the mRNA message itself in the form of cis-regulatory elements bearing particular primary sequences and/or secondary-structures. Such control elements are operated by RNA-binding proteins (RBPs) and/or miRNA-containing complexes. Based on the large number of RBPs and miRNAs encoded in metazoan genomes, their complex developmental expression and that specific RBP and miRNA interactions with mRNAs can lead to distinct degradation rates, I propose that developmental gene expression is shaped by a complex 'mRNA degradation code' with high information capacity. Localised cellular events involving the modification of RBP and/or miRNA target sequences in mRNAs by alternative polyadenylation added to the activation of specific RBP and miRNA activities via cell signalling are predicted to further expand the capacity of the mRNA degradation code by coupling it to dynamic events experienced by cells at specific spatiotemporal coordinates within the developing embryo.
Asunto(s)
Regulación de la Expresión Génica , Estabilidad del ARN , ARN Mensajero/metabolismo , Animales , Humanos , MicroARNs/genética , MicroARNs/metabolismo , ARN Mensajero/genética , Proteínas de Unión al ARN/metabolismoRESUMEN
Hox genes encode a family of transcriptional regulators that operate differential developmental programs along the anteroposterior axis of bilateral animals. Regulatory changes affecting Hox gene expression are believed to have been crucial for the evolution of animal body plans. In Drosophila melanogaster, Hox expression is post-transcriptionally regulated by microRNAs (miRNAs) acting on target sites located in the 3' untranslated regions (3'UTRs) of Hox mRNAs. Notably, recent work has shown that during D. melanogaster development Hox genes produce mRNAs with variable 3'UTRs (short and long forms) in different sets of tissues as a result of alternative polyadenylation; importantly, Hox short and long 3'UTRs contain very different target sites for miRNAs. Here, we use a computational approach to explore the evolution of Hox 3'UTRs treated with especial regard to miRNA regulation. Our work is focused on the 12 Drosophila species for which genomic sequences are available and shows, first, that alternative polyadenylation of Hox transcripts is a feature shared by all drosophilids tested in the study. Second, that the regulatory impact of miRNAs is evolving very fast within the Drosophila group. Third, that in contrast to the low degree of primary sequence conservation, Hox 3'UTR regions within the group show very similar RNA topology indicating that RNA structure is under strong selective pressure. Finally, we also demonstrate that Hox alternative polyadenylation can remodel the control regions seen by miRNAs by at least two mechanisms: via adding new cis-regulatory sequences-in the form of miRNA target sites-to short 3'UTR forms as well as by modifying the regulatory impact of miRNA target sites in short 3'UTR forms through changes in RNA secondary structure caused by the use of distal polyadenylation signals.
Asunto(s)
Regiones no Traducidas 3'/genética , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Genes Homeobox/genética , Proteínas de Homeodominio/genética , MicroARNs/genética , Procesamiento Postranscripcional del ARN , Factores de Transcripción/genética , Animales , Biología Computacional , Secuencia Conservada , Evolución Molecular , Regulación de la Expresión Génica , Genoma de los Insectos , Poliadenilación/genética , ARN Mensajero/genéticaRESUMEN
Nonsense-mediated RNA decay (NMD) is a surveillance mechanism that degrades transcripts containing nonsense mutations, preventing the translation of truncated proteins. NMD also regulates the levels of many endogenous mRNAs. While the mechanism of NMD is gradually understood, its physiological role remains largely unknown. The core NMD genes upf1 and upf2 are essential in several organisms, which may reflect an important developmental role for NMD. Alternatively, the lethality of these mutants might arise from their function in NMD-independent processes. To analyze the developmental importance of NMD, we studied Drosophila mutants of the other core NMD gene, upf3. We compare the resulting upf3 phenotype with those defects observed in upf1 and upf2 loss-of-function mutants, as well as with flies expressing a mutant Upf2 protein unable to bind Upf3. Our results show that Upf3 is an NMD effector in the fly but, unlike Upf1 and Upf2, plays a peripheral role in the degradation of most NMD targets and is not required for development or viability. Furthermore, Upf1 and Upf2 loss-of-function inhibits cell growth and induces apoptosis through a Upf3-independent pathway. Accordingly, disruption of Upf2-Upf1 interaction causes death, while the Upf2-Upf3 complex is dispensable for viability. Our findings suggest that NMD is essential for cell growth and animal development, and that the lethality of upf1 and upf2 mutants is not due to disrupting their roles during NMD-independent processes, but to their function in the degradation of specific mRNAs by the NMD pathway. Furthermore, our results show that Upf3 is not always essential in NMD.
Asunto(s)
Codón sin Sentido/metabolismo , Proteínas de Drosophila/fisiología , Drosophila melanogaster/fisiología , Genes Letales , Procesamiento Postranscripcional del ARN/genética , Estabilidad del ARN/genética , Alelos , Animales , Proliferación Celular , Codón sin Sentido/genética , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Drosophila melanogaster/crecimiento & desarrolloRESUMEN
Although most metazoan genes undergo alternative splicing, the functional relevance of the majority of alternative splicing products is still unknown. Here we explore this problem in the Drosophila Hox gene Ultrabithorax (Ubx). Ubx produces a family of six protein isoforms through alternative splicing. To investigate the functional specificity of the Ubx isoforms, we studied their role during the formation of the Drosophila halteres, small dorsal appendages that are essential for normal flight. Our work shows that isoform Ia, which is encoded by all Ubx exons, is more efficient than isoform IVa, which lacks the amino acids coded by two small exons, in controlling haltere development and regulating Ubx downstream targets. However, our experiments also demonstrate that the functional differences among the Ubx isoforms can be compensated for by increasing the expression levels of the less efficient form. The analysis of the DNA-binding profiles of Ubx isoforms to a natural Ubx target, spalt, shows no major differences in isoform DNA-binding activities, suggesting that alternative splicing might primarily affect the regulatory capacity of the isoforms rather than their DNA-binding patterns. Our results suggest that to obtain distinct functional outputs during normal development genes must integrate the generation of qualitative differences by alternative splicing to quantitative processes affecting isoform protein expression levels.
Asunto(s)
Proteínas de Drosophila/metabolismo , Proteínas de Homeodominio/metabolismo , ARN/metabolismo , Factores de Transcripción/metabolismo , Empalme Alternativo/genética , Empalme Alternativo/fisiología , Animales , Western Blotting , Drosophila , Proteínas de Drosophila/genética , Regulación del Desarrollo de la Expresión Génica/genética , Regulación del Desarrollo de la Expresión Génica/fisiología , Proteínas de Homeodominio/genética , Fenotipo , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , ARN/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factores de Transcripción/genéticaRESUMEN
BACKGROUND: The modulation of mRNA levels across tissues and time is key for the establishment and operation of the developmental programs that transform the fertilized egg into a fully formed embryo. Although the developmental mechanisms leading to differential mRNA synthesis are heavily investigated, comparatively little attention is given to the processes of mRNA degradation and how these relate to the molecular programs controlling development. RESULTS: Here we combine timed collection of Drosophila embryos and unfertilized eggs with genome-wide microarray technology to determine the degradation patterns of all mRNAs present during early fruit fly development. Our work studies the kinetics of mRNA decay, the contributions of maternally and zygotically encoded factors to mRNA degradation, and the ways in which mRNA decay profiles relate to gene function, mRNA localization patterns, translation rates and protein turnover. We also detect cis-regulatory sequences enriched in transcripts with common degradation patterns and propose several proteins and microRNAs as developmental regulators of mRNA decay during early fruit fly development. Finally, we experimentally validate the effects of a subset of cis-regulatory sequences and trans-regulators in vivo. CONCLUSIONS: Our work advances the current understanding of the processes controlling mRNA degradation during early Drosophila development, taking us one step closer to the understanding of mRNA decay processes in all animals. Our data also provide a valuable resource for further experimental and computational studies investigating the process of mRNA decay.