RESUMO
The sequence-specific RNA-binding protein Pumilio (Pum) controls Drosophila development; however, the network of mRNAs that it regulates remains incompletely characterized. In this study, we use knockdown and knockout approaches coupled with RNA-seq to measure the impact of Pum on the transcriptome of Drosophila cells in culture. We also use an improved RNA coimmunoprecipitation method to identify Pum-bound mRNAs in Drosophila embryos. Integration of these data sets with the locations of Pum-binding motifs across the transcriptome reveals novel direct Pum target genes involved in neural, muscle, wing, and germ cell development and in cellular proliferation. These genes include components of Wnt, TGF-ß, MAPK/ERK, and Notch signaling pathways, DNA replication, and lipid metabolism. We identify the mRNAs regulated by the CCR4-NOT deadenylase complex, a key factor in Pum-mediated repression, and observe concordant regulation of Pum:CCR4-NOT target mRNAs. Computational modeling reveals that Pum binding, binding site number, clustering, and sequence context are important determinants of regulation. In contrast, we show that the responses of direct mRNA targets to Pum-mediated repression are not influenced by the content of optimal synonymous codons. Moreover, contrary to a prevailing model, we do not detect a role for CCR4-NOT in the degradation of mRNAs with low codon optimality. Together, the results of this work provide new insights into the Pum regulatory network and mechanisms and the parameters that influence the efficacy of Pum-mediated regulation.
Assuntos
Proteínas de Drosophila , Proteínas de Ligação a RNA , Transcriptoma , Animais , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Proteínas de Ligação a RNA/metabolismo , Proteínas de Ligação a RNA/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Ribonucleases/metabolismo , Ribonucleases/genética , Regulação da Expressão Gênica no Desenvolvimento , Sítios de Ligação , Ligação Proteica , Drosophila/genética , Drosophila/metabolismoRESUMO
Small non-coding RNAs called piRNAs serve as guides for an adaptable immune system that represses transposable elements in germ cells of Metazoa. In Drosophila the RDC complex, composed of Rhino, Deadlock and Cutoff (Cuff) bind chromatin of dual-strand piRNA clusters, special genomic regions, which encode piRNA precursors. The RDC complex is required for transcription of piRNA precursors, though the mechanism by which it licenses transcription remained unknown. Here, we show that Cuff prevents premature termination of RNA polymerase II. Cuff prevents cleavage of nascent RNA at poly(A) sites by interfering with recruitment of the cleavage and polyadenylation specificity factor (CPSF) complex. Cuff also protects processed transcripts from degradation by the exonuclease Rat1. Our work reveals a conceptually different mechanism of transcriptional enhancement. In contrast to other factors that regulate termination by binding to specific signals on nascent RNA, the RDC complex inhibits termination in a chromatin-dependent and sequence-independent manner.
Assuntos
Proteínas de Drosophila/metabolismo , Drosophila melanogaster/enzimologia , RNA Polimerase II/metabolismo , RNA Interferente Pequeno/biossíntese , Proteínas de Ligação a RNA/metabolismo , Transcrição Gênica , Adenosina/metabolismo , Animais , Animais Geneticamente Modificados , Sítios de Ligação , Cromatina/metabolismo , Proteínas Cromossômicas não Histona/metabolismo , Fator de Especificidade de Clivagem e Poliadenilação/metabolismo , Biologia Computacional , Bases de Dados Genéticas , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Exorribonucleases/metabolismo , Genes Reporter , Proteínas Associadas aos Microtúbulos/metabolismo , Complexos Multiproteicos , Polímeros/metabolismo , Ligação Proteica , Estabilidade de RNA , RNA Interferente Pequeno/genética , Proteínas de Ligação a RNA/genética , Terminação da Transcrição GenéticaRESUMO
Localization of oskar mRNA includes two distinct phases: transport from nurse cells to the oocyte, a process typically accompanied by cortical anchoring in the oocyte, followed by posterior localization within the oocyte. Signals within the oskar 3' UTR directing transport are individually weak, a feature previously hypothesized to facilitate exchange between the different localization machineries. We show that alteration of the SL2a stem-loop structure containing the oskar transport and anchoring signal (TAS) removes an inhibitory effect such that in vitro binding by the RNA transport factor, Egalitarian, is elevated as is in vivo transport from the nurse cells into the oocyte. Cortical anchoring within the oocyte is also enhanced, interfering with posterior localization. We also show that mutation of Staufen recognized structures (SRSs), predicted binding sites for Staufen, disrupts posterior localization of oskar mRNA just as in staufen mutants. Two SRSs in SL2a, one overlapping the Egalitarian binding site, are inferred to mediate Staufen-dependent inhibition of TAS anchoring activity, thereby promoting posterior localization. The other three SRSs in the oskar 3' UTR are also required for posterior localization, including two located distant from any known transport signal. Staufen, thus, plays multiple roles in localization of oskar mRNA.
Assuntos
Proteínas de Drosophila/genética , Oócitos/crescimento & desenvolvimento , Proteínas de Ligação a RNA/genética , Animais , Proteínas de Drosophila/ultraestrutura , Drosophila melanogaster/genética , Drosophila melanogaster/crescimento & desenvolvimento , Sequências Repetidas Invertidas/genética , Mutação/genética , Proteínas de Ligação a RNA/ultraestruturaRESUMO
Mutations in RanBP2 (also known as Nup358), one of the main components of the cytoplasmic filaments of the nuclear pore complex, contribute to the overproduction of acute necrotizing encephalopathy (ANE1)-associated cytokines. Here we report that RanBP2 represses the translation of the interleukin 6 (IL6) mRNA, which encodes a cytokine that is aberrantly up-regulated in ANE1. Our data indicates that soon after its production, the IL6 messenger ribonucleoprotein (mRNP) recruits Argonautes bound to let-7 microRNA. After this mRNP is exported to the cytosol, RanBP2 sumoylates mRNP-associated Argonautes, thereby stabilizing them and enforcing mRNA silencing. Collectively, these results support a model whereby RanBP2 promotes an mRNP remodelling event that is critical for the miRNA-mediated suppression of clinically relevant mRNAs, such as IL6.
Assuntos
Proteínas Argonautas/genética , Fatores de Iniciação em Eucariotos/genética , Regulação da Expressão Gênica , MicroRNAs/genética , Chaperonas Moleculares/genética , Complexo de Proteínas Formadoras de Poros Nucleares/genética , Regiões 3' não Traduzidas/genética , Proteínas Argonautas/metabolismo , Linhagem Celular Tumoral , Fatores de Iniciação em Eucariotos/metabolismo , Células HEK293 , Humanos , Interleucina-6/genética , Interleucina-6/metabolismo , MicroRNAs/metabolismo , Chaperonas Moleculares/metabolismo , Mutação , Complexo de Proteínas Formadoras de Poros Nucleares/metabolismo , Pancreatite Necrosante Aguda/genética , Pancreatite Necrosante Aguda/metabolismo , Ligação Proteica , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , SumoilaçãoRESUMO
From fly to mammals, the Smaug/Samd4 family of prion-like RNA-binding proteins control gene expression by destabilizing and/or repressing the translation of numerous target transcripts. However, the regulation of its activity remains poorly understood. We show that Smaug's protein levels and mRNA repressive activity are downregulated by Hedgehog signaling in tissue culture cells. These effects rely on the interaction of Smaug with the G-protein coupled receptor Smoothened, which promotes the phosphorylation of Smaug by recruiting the kinase Fused. The activation of Fused and its binding to Smaug are sufficient to suppress its ability to form cytosolic bodies and to antagonize its negative effects on endogenous targets. Importantly, we demonstrate in vivo that HH reduces the levels of smaug mRNA and increases the level of several mRNAs downregulated by Smaug. Finally, we show that Smaug acts as a positive regulator of Hedgehog signaling during wing morphogenesis. These data constitute the first evidence for a post-translational regulation of Smaug and reveal that the fate of several mRNAs bound to Smaug is modulated by a major signaling pathway.
Assuntos
Proteínas de Drosophila , Drosophila , Animais , Drosophila/genética , Drosophila/metabolismo , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Proteínas Hedgehog/genética , Proteínas Hedgehog/metabolismo , Proteínas de Ligação a RNA/genética , Receptores Acoplados a Proteínas G/genética , Proteínas Repressoras/metabolismo , Receptor Smoothened/genéticaRESUMO
Post-transcriptional regulation of mRNAs plays an essential role in the control of gene expression. mRNAs are regulated in ribonucleoprotein (RNP) complexes by RNA-binding proteins (RBPs) along with associated protein and noncoding RNA (ncRNA) cofactors. A global understanding of post-transcriptional control in any cell type requires identification of the components of all of its RNP complexes. We have previously shown that these complexes can be purified by immunoprecipitation using anti-RBP synthetic antibodies produced by phage display. To develop the large number of synthetic antibodies required for a global analysis of RNP complex composition, we have established a pipeline that combines (i) a computationally aided strategy for design of antigens located outside of annotated domains, (ii) high-throughput antigen expression and purification in Escherichia coli, and (iii) high-throughput antibody selection and screening. Using this pipeline, we have produced 279 antibodies against 61 different protein components of Drosophila melanogaster RNPs. Together with those produced in our low-throughput efforts, we have a panel of 311 antibodies for 67 RNP complex proteins. Tests of a subset of our antibodies demonstrated that 89% immunoprecipitate their endogenous target from embryo lysate. This panel of antibodies will serve as a resource for global studies of RNP complexes in Drosophila. Furthermore, our high-throughput pipeline permits efficient production of synthetic antibodies against any large set of proteins.
Assuntos
Anticorpos/química , Proteínas de Drosophila/imunologia , Ribonucleoproteínas/imunologia , Sequência de Aminoácidos , Animais , Anticorpos/metabolismo , Antígenos/imunologia , Antígenos/isolamento & purificação , Western Blotting , Regiões Determinantes de Complementaridade , Proteínas de Drosophila/isolamento & purificação , Drosophila melanogaster , Ensaio de Imunoadsorção Enzimática , Escherichia coli , Imunoprecipitação , Dados de Sequência Molecular , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Ribonucleoproteínas/isolamento & purificaçãoRESUMO
Here, we have asked about post-transcriptional mechanisms regulating murine developmental neurogenesis, focusing upon the RNA-binding proteins Smaug2 and Nanos1. We identify, in embryonic neural precursors of the murine cortex, a Smaug2 protein/nanos1 mRNA complex that is present in cytoplasmic granules with the translational repression proteins Dcp1 and 4E-T. We show that Smaug2 inhibits and Nanos1 promotes neurogenesis, with Smaug2 knockdown enhancing neurogenesis and depleting precursors, and Nanos1 knockdown inhibiting neurogenesis and maintaining precursors. Moreover, we show that Smaug2 likely regulates neurogenesis by silencing nanos1 mRNA. Specifically, Smaug2 knockdown inappropriately increases Nanos1 protein, and the Smaug2 knockdown-mediated neurogenesis is rescued by preventing this increase. Thus, Smaug2 and Nanos1 function as a bimodal translational repression switch to control neurogenesis, with Smaug2 acting in transcriptionally primed precursors to silence mRNAs important for neurogenesis, including nanos1 mRNA, and Nanos1 acting during the transition to neurons to repress the precursor state. SIGNIFICANCE STATEMENT: The mechanisms instructing neural stem cells to generate the appropriate progeny are still poorly understood. Here, we show that the RNA-binding proteins Smaug2 and Nanos1 are critical regulators of this balance and provide evidence supporting the idea that neural precursors are transcriptionally primed to generate neurons but translational regulation maintains these precursors in a stem cell state until the appropriate developmental time.
Assuntos
Diferenciação Celular/fisiologia , Córtex Cerebral/fisiologia , Células-Tronco Neurais/fisiologia , Neurogênese/fisiologia , Proteínas de Ligação a RNA/fisiologia , Proteínas Repressoras/fisiologia , Animais , Células Cultivadas , Córtex Cerebral/citologia , Córtex Cerebral/embriologia , Feminino , Masculino , Mamíferos , Camundongos , Biossíntese de Proteínas/fisiologiaRESUMO
Argonaute (Ago) proteins are typically recruited to target messenger RNAs via an associated small RNA such as a microRNA (miRNA). Here, we describe a new mechanism of Ago recruitment through the Drosophila Smaug RNA-binding protein. We show that Smaug interacts with the Ago1 protein, and that Ago1 interacts with and is required for the translational repression of the Smaug target, nanos mRNA. The Ago1/nanos mRNA interaction does not require a miRNA, but it does require Smaug. Taken together, our data suggest a model whereby Smaug directly recruits Ago1 to nanos mRNA in a miRNA-independent manner, thereby repressing translation.
Assuntos
Proteínas Argonautas/genética , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila/genética , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Proteínas Repressoras/genética , Animais , Proteínas Argonautas/metabolismo , Drosophila/metabolismo , Feminino , Regulação da Expressão Gênica , MicroRNAs , Ligação Proteica , RNA Mensageiro/genética , Proteínas Repressoras/metabolismo , Transdução de SinaisRESUMO
Despite studies that have investigated the interactions of double-stranded RNA-binding proteins like Staufen with RNA in vitro, how they achieve target specificity in vivo remains uncertain. We performed RNA co-immunoprecipitations followed by microarray analysis to identify Staufen-associated mRNAs in early Drosophila embryos. Analysis of the localization and functions of these transcripts revealed a number of potentially novel roles for Staufen. Using computational methods, we identified two sequence features that distinguish Staufen's target transcripts from non-targets. First, these Drosophila transcripts, as well as those human transcripts bound by human Staufen1 and 2, have 3' untranslated regions (UTRs) that are 3-4-fold longer than unbound transcripts. Second, the 3'UTRs of Staufen-bound transcripts are highly enriched for three types of secondary structures. These structures map with high precision to previously identified Staufen-binding regions in Drosophila bicoid and human ARF1 3'UTRs. Our results provide the first systematic genome-wide analysis showing how a double-stranded RNA-binding protein achieves target specificity.
Assuntos
Regiões 3' não Traduzidas , Proteínas do Citoesqueleto/metabolismo , Proteínas de Drosophila/metabolismo , Proteínas de Ligação a RNA/metabolismo , Animais , Drosophila/embriologia , Drosophila/genética , Genoma de Inseto , Humanos , Conformação de Ácido Nucleico , RNA de Cadeia Dupla/química , RNA de Cadeia Dupla/metabolismo , RNA Mensageiro/análise , RNA Mensageiro/metabolismoRESUMO
Complexes that control mRNA stability and translation promote timely cell-state transitions during differentiation by ensuring appropriate expression patterns of key developmental regulators. The Drosophila RNA-binding protein Brain tumor (Brat) promotes degradation of target transcripts during the maternal-to-zygotic transition in syncytial embryos and in uncommitted intermediate neural progenitors (immature INPs). We identified Ubiquitin-specific protease 5 (Usp5) as a Brat interactor essential for the degradation of Brat target mRNAs in both cell types. Usp5 promotes Brat-dedadenylase pre-complex assembly in mitotic neural stem cells (neuroblasts) by bridging Brat and the scaffolding components of deadenylase complexes lacking their catalytic subunits. The adaptor protein Miranda binds the RNA-binding domain of Brat, limiting its ability to bind target mRNAs in mitotic neuroblasts. Cortical displacement of Miranda activates Brat-mediated mRNA decay in immature INPs. We propose that the assembly of an enzymatically inactive and RNA-binding-deficient pre-complex poises mRNA degradation machineries for rapid activation driving timely developmental transitions.
RESUMO
During Drosophila oogenesis, the Oskar (OSK) RNA binding protein (RBP) determines the amount of germ plasm that assembles at the posterior pole of the oocyte. Here, we identify mechanisms that subsequently regulate germ plasm assembly in the early embryo. We show that the Smaug (SMG) RBP is transported into the germ plasm of the early embryo where it accumulates in the germ granules. SMG binds to and represses translation of the osk messenger RNA (mRNA) as well as the bruno 1 (bru1) mRNA, which encodes an RBP that we show promotes germ plasm production. Loss of SMG or mutation of SMG's binding sites in the osk or bru1 mRNA results in excess translation of these transcripts in the germ plasm, accumulation of excess germ plasm, and budding of excess primordial germ cells (PGCs). Therefore, SMG triggers a posttranscriptional regulatory pathway that attenuates the amount of germ plasm in embryos to modulate the number of PGCs.
Assuntos
Drosophila , Lagartos , Animais , Citoplasma , Células Germinativas , RNA Mensageiro/genética , Contagem de CélulasRESUMO
During Drosophila oogenesis, the Oskar (OSK) RNA-binding protein (RBP) determines the amount of germ plasm that assembles at the posterior pole of the oocyte. Here we identify the mechanisms that regulate the osk mRNA in the early embryo. We show that the Smaug (SMG) RBP is transported into the germ plasm of the early embryo where it accumulates in the germ granules. SMG binds to and represses translation of the osk mRNA itself as well as the bruno 1 (bru1) mRNA, which encodes an RBP that we show promotes germ plasm production. Loss of SMG or mutation of SMG's binding sites in the osk or bru1 mRNAs results in ectopic translation of these transcripts in the germ plasm and excess PGCs. SMG therefore triggers a post-transcriptional regulatory pathway that attenuates germ plasm synthesis in embryos, thus modulating the number of PGCs.
RESUMO
The sequence-specific RNA-binding protein Pumilio controls development of Drosophila; however, the network of mRNAs that it regulates remains incompletely characterized. In this study, we utilize knockdown and knockout approaches coupled with RNA-Seq to measure the impact of Pumilio on the transcriptome of Drosophila cells. We also used an improved RNA co-immunoprecipitation method to identify Pumilio bound mRNAs in Drosophila embryos. Integration of these datasets with the content of Pumilio binding motifs across the transcriptome revealed novel direct Pumilio target genes involved in neural, muscle, wing, and germ cell development, and cellular proliferation. These genes include components of Wnt, TGF-beta, MAPK/ERK, and Notch signaling pathways, DNA replication, and lipid metabolism. Additionally, we identified the mRNAs regulated by the CCR4-NOT deadenylase complex, a key factor in Pumilio-mediated repression, and observed concordant regulation of Pumilio:CCR4-NOT target mRNAs. Computational modeling revealed that Pumilio binding, binding site number, density, and sequence context are important determinants of regulation. Moreover, the content of optimal synonymous codons in target mRNAs exhibits a striking functional relationship to Pumilio and CCR4-NOT regulation, indicating that the inherent translation efficiency and stability of the mRNA modulates their response to these trans-acting regulatory factors. Together, the results of this work provide new insights into the Pumilio regulatory network and mechanisms, and the parameters that influence the efficacy of Pumilio-mediated regulation.
RESUMO
In animals, egg activation triggers a cascade of posttranscriptional events that act on maternally synthesized RNAs. We show that, in Drosophila, the PAN GU (PNG) kinase sits near the top of this cascade, triggering translation of SMAUG (SMG), a multifunctional posttranscriptional regulator conserved from yeast to humans. Although PNG is required for cytoplasmic polyadenylation of smg mRNA, it regulates translation via mechanisms that are independent of its effects on the poly(A) tail. Analyses of mutants suggest that PNG relieves translational repression by PUMILIO (PUM) and one or more additional factors, which act in parallel through the smg mRNA's 3' untranslated region (UTR). Microarray-based gene expression profiling shows that SMG is a major regulator of maternal transcript destabilization. SMG-dependent mRNAs are enriched for gene ontology annotations for function in the cell cycle, suggesting a possible causal relationship between failure to eliminate these transcripts and the cell cycle defects in smg mutants.
Assuntos
Proteínas de Drosophila/metabolismo , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Biossíntese de Proteínas , Proteínas Serina-Treonina Quinases/metabolismo , Estabilidade de RNA , RNA Mensageiro Estocado/metabolismo , Proteínas de Ligação a RNA/metabolismo , Proteínas Repressoras/metabolismo , Regiões 3' não Traduzidas/metabolismo , Animais , Biologia Computacional , Citoplasma/metabolismo , Proteínas de Drosophila/genética , Drosophila melanogaster/citologia , Drosophila melanogaster/embriologia , Embrião não Mamífero/citologia , Feminino , Perfilação da Expressão Gênica , Regulação da Expressão Gênica no Desenvolvimento , Proteínas de Choque Térmico/genética , Análise em Microsséries , Modelos Genéticos , Mutação/genética , Óvulo , Poliadenilação , Proteínas Serina-Treonina Quinases/genética , Proteínas de Ligação a RNA/genética , Proteínas Repressoras/genéticaRESUMO
The SAM domain of the Saccharomyces cerevisiae post-transcriptional regulator Vts1p epitomizes a subfamily of SAM domains conserved from yeast to humans that function as sequence-specific RNA-binding domains. Here we report the 2.0-A X-ray structure of the Vts1p SAM domain bound to a high-affinity RNA ligand. Specificity of RNA binding arises from the association of a guanosine loop base with a shallow pocket on the SAM domain and from multiple SAM domain contacts to the unique backbone structure of the loop, defined in part by a nonplanar base pair within the loop. We have validated NNF1 as an endogenous target of Vts1p among 79 transcripts that copurify with Vts1p. Bioinformatic analysis of these mRNAs demonstrates that the RNA-binding specificity of Vts1p in vivo is probably more stringent than that of the isolated SAM domain in vitro.
Assuntos
Conformação de Ácido Nucleico , RNA Fúngico/genética , RNA Fúngico/metabolismo , Proteínas de Ligação a RNA/química , Proteínas de Ligação a RNA/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/metabolismo , Pareamento de Bases , Sítios de Ligação , Cristalografia por Raios X , Internet , Modelos Moleculares , Ligação Proteica , Estrutura Terciária de Proteína , RNA Fúngico/química , Proteínas de Ligação a RNA/genética , Elementos de Resposta/genética , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Especificidade por SubstratoRESUMO
The Smaug family of sequence-specific RNA binding proteins regulates mRNA translation and degradation by binding to consensus stem-loop structures in target mRNAs. Vts1p is a member of the Smaug protein family that regulates the stability of target transcripts in Saccharomyces cerevisiae. Here we focus on the mechanism of Vts1p-mediated mRNA decay. Using RNA reporters that recapitulate Vts1p-mediated decay in vivo, we demonstrate that Vts1p stimulates mRNA degradation through deadenylation mediated by the Ccr4p-Pop2p-Not deadenylase complex. We also show that Vts1p interacts with the Ccr4p-Pop2p-Not complex suggesting that Vts1p recruits the Ccr4p-Pop2p-Not deadenylase complex to target mRNAs, resulting in transcript decay. Following deadenylation Vts1p target transcripts are decapped and subsequently degraded by the 5'-to-3' exonuclease Xrn1p. Decapping and 5'-to-3' decay is thought to occur in foci known as P-bodies, and we provide evidence that Vts1p function may involve P-bodies. Taken together with previous work, these data suggest that Smaug family members employ a conserved mechanism to induce transcript degradation that involves recruitment of the Ccr4-Pop2-Not deadenylase to target mRNAs.
Assuntos
Ribonucleases/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Complexos Multienzimáticos/metabolismo , Estabilidade de RNA , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA , Saccharomyces cerevisiaeRESUMO
Actinobacteria produce antibacterial and antifungal specialized metabolites. Many insects harbour actinobacteria on their bodies or in their nests and use these metabolites for protection. However, some actinobacteria produce metabolites that are toxic to insects and the evolutionary relevance of this toxicity is unknown. Here we explore chemical interactions between streptomycetes and the fruit fly Drosophila melanogaster. We find that many streptomycetes produce specialized metabolites that have potent larvicidal effects against the fly; larvae that ingest spores of these species die. The mechanism of toxicity is specific to the bacterium's chemical arsenal: cosmomycin D producing bacteria induce a cell death-like response in the larval digestive tract; avermectin producing bacteria induce paralysis. Furthermore, low concentrations of volatile terpenes like 2-methylisoborneol that are produced by streptomycetes attract fruit flies such that they preferentially deposit their eggs on contaminated food sources. The resulting larvae are killed during growth and development. The phenomenon of volatile-mediated attraction and specialized metabolite toxicity suggests that some streptomycetes pose an evolutionary risk to insects in nature.
Assuntos
Bactérias/metabolismo , Drosophila melanogaster/citologia , Drosophila melanogaster/microbiologia , Actinobacteria/fisiologia , Animais , Antraciclinas/farmacologia , Antibacterianos/química , Antibacterianos/farmacologia , Canfanos/toxicidade , Morte Celular/efeitos dos fármacos , Drosophila melanogaster/efeitos dos fármacos , Larva/efeitos dos fármacos , Larva/microbiologia , Metaboloma , Esporos Bacterianos/metabolismo , Esporos Bacterianos/fisiologia , Streptomyces/fisiologia , Análise de Sobrevida , Compostos Orgânicos Voláteis/farmacologiaRESUMO
In animal embryos, the maternal-to-zygotic transition (MZT) hands developmental control from maternal to zygotic gene products. We show that the maternal proteome represents more than half of the protein-coding capacity of Drosophila melanogaster's genome, and that 2% of this proteome is rapidly degraded during the MZT. Cleared proteins include the post-transcriptional repressors Cup, Trailer hitch (TRAL), Maternal expression at 31B (ME31B), and Smaug (SMG). Although the ubiquitin-proteasome system is necessary for clearance of these repressors, distinct E3 ligase complexes target them: the C-terminal to Lis1 Homology (CTLH) complex targets Cup, TRAL, and ME31B for degradation early in the MZT and the Skp/Cullin/F-box-containing (SCF) complex targets SMG at the end of the MZT. Deleting the C-terminal 233 amino acids of SMG abrogates F-box protein interaction and confers immunity to degradation. Persistent SMG downregulates zygotic re-expression of mRNAs whose maternal contribution is degraded by SMG. Thus, clearance of SMG permits an orderly MZT.
Assuntos
Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Regulação da Expressão Gênica no Desenvolvimento , Proteínas Repressoras/genética , Transcrição Gênica , Zigoto/metabolismo , Animais , Regulação para Baixo/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/embriologia , Embrião não Mamífero/metabolismo , Desenvolvimento Embrionário/genética , Feminino , Complexo de Endopeptidases do Proteassoma/metabolismo , Ligação Proteica , Biossíntese de Proteínas/genética , Subunidades Proteicas/metabolismo , Proteólise , Proteoma/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteínas Repressoras/metabolismo , Ribonucleoproteínas/metabolismo , Fatores de Tempo , Transcriptoma/genética , Ubiquitina/metabolismoRESUMO
G3BP RNA-binding proteins are important components of stress granules (SGs). Here, we analyze the role of the Drosophila G3BP Rasputin (RIN) in unstressed cells, where RIN is not SG associated. Immunoprecipitation followed by microarray analysis identifies over 550 mRNAs that copurify with RIN. The mRNAs found in SGs are long and translationally silent. In contrast, we find that RIN-bound mRNAs, which encode core components of the transcription, splicing, and translation machinery, are short, stable, and highly translated. We show that RIN is associated with polysomes and provide evidence for a direct role for RIN and its human homologs in stabilizing and upregulating the translation of their target mRNAs. We propose that when cells are stressed, the resulting incorporation of RIN/G3BPs into SGs sequesters them away from their short target mRNAs. This would downregulate the expression of these transcripts, even though they are not incorporated into stress granules.