RESUMO
Despite recent advances in developmental biology, and the sequencing and annotation of genomes, key questions regarding the organisation of cells into embryos remain. One possibility is that uncharacterised genes having nonstandard coding arrangements and functions could provide some of the answers. Here we present the characterisation of tarsal-less (tal), a new type of noncanonical gene that had been previously classified as a putative noncoding RNA. We show that tal controls gene expression and tissue folding in Drosophila, thus acting as a link between patterning and morphogenesis. tal function is mediated by several 33-nucleotide-long open reading frames (ORFs), which are translated into 11-amino-acid-long peptides. These are the shortest functional ORFs described to date, and therefore tal defines two novel paradigms in eukaryotic coding genes: the existence of short, unprocessed peptides with key biological functions, and their arrangement in polycistronic messengers. Our discovery of tal-related short ORFs in other species defines an ancient and noncanonical gene family in metazoans that represents a new class of eukaryotic genes. Our results open a new avenue for the annotation and functional analysis of genes and sequenced genomes, in which thousands of short ORFs are still uncharacterised.
Assuntos
Proteínas de Drosophila/genética , Extremidades/embriologia , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Genes de Insetos/fisiologia , Fases de Leitura Aberta/fisiologia , Transaldolase/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Drosophila melanogaster/embriologia , Dados de Sequência Molecular , Biossíntese de ProteínasRESUMO
In human, the myeloid leukemia factor 1 (hMLF1) has been shown to be involved in acute leukemia, and mlf related genes are present in many animals. Despite their extensive representation and their good conservation, very little is understood about their function. In Drosophila, dMLF physically interacts with both the transcription regulatory factor DREF and an antagonist of the Hedgehog pathway, Suppressor of Fused, whose over-expression in the fly suppresses the toxicity induced by polyglutamine. No connection between these data has, however, been established. Here, we show that dmlf is widely and dynamically expressed during fly development. We isolated and analyzed the first dmlf mutants: embryos lacking maternal dmlf product have a low viability with no specific defect, and dmlf(-)- adults display weak phenotypes. We monitored dMLF subcellular localization in the fly and cultured cells. We were able to show that, although generally nuclear, dMLF can also be cytoplasmic, depending on the developmental context. Furthermore, two differently spliced variants of dMLF display differential subcellular localization, allowing the identification of regions of dMLF potentially important for its localization. Finally, we demonstrate that dMLF can act developmentally and postdevelopmentally to suppress neurodegeneration and premature aging in a cerebellar ataxia model.
Assuntos
Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila/embriologia , Drosophila/genética , Processamento Alternativo , Sequência de Aminoácidos , Animais , Animais Geneticamente Modificados , Núcleo Celular/metabolismo , Sequência Conservada , Citoplasma/metabolismo , Drosophila/metabolismo , Proteínas de Drosophila/química , Embrião não Mamífero , Olho/metabolismo , Olho/ultraestrutura , Regulação da Expressão Gênica no Desenvolvimento , Imuno-Histoquímica , Dados de Sequência Molecular , Mutação , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Proteínas Repressoras/metabolismo , Análise de Sequência de DNA , Homologia de Sequência de Aminoácidos , Frações Subcelulares/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
In this study we describe a novel Drosophila protein Jupiter, which shares properties with several structural microtubule-associated proteins (MAPs) including TAU, MAP2, MAP4. Jupiter is a soluble unfolded molecule with the high net positive charge, rich in Glycine. It possesses two degenerated repeats around the sequence PPGG, separated by a Serine-rich region. Jupiter associates with microtubules in vitro and, fused with the green fluorescent protein (GFP), is an excellent marker to follow microtubule dynamics in vivo. In a jupiter transgenic Drosophila strain generated by the "protein-trap" technique, Jupiter:GFP fusion protein localizes to the microtubule network through the cell cycle at the different stages of development. We found particularly high Jupiter:GFP concentrations in the young embryo, larval nervous system, precursors of eye photoreceptors and adult ovary. Moreover, from jupiter:gfp embryos we have established two permanent cell lines presenting strongly fluorescent microtubules during the whole cell cycle. In these cells, the distribution of the Jupiter:GFP fusion protein reproduces microtubule behavior upon treatment by the drugs colchicine and taxol. The jupiter cell lines and fly strain should be of wide interest for biologists interested in in vivo analysis of microtubule dynamics.
Assuntos
Proteínas de Drosophila/análise , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Proteínas dos Microtúbulos/análise , Proteínas dos Microtúbulos/metabolismo , Microtúbulos/química , Sequência de Aminoácidos , Animais , Proteínas de Drosophila/genética , Drosophila melanogaster/citologia , Drosophila melanogaster/genética , Embrião não Mamífero/química , Embrião não Mamífero/citologia , Genes de Insetos , Proteínas de Fluorescência Verde/análise , Proteínas de Fluorescência Verde/genética , Microscopia de Fluorescência , Proteínas dos Microtúbulos/genética , Microtúbulos/metabolismo , Dados de Sequência Molecular , Proteínas Recombinantes de Fusão/análise , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismoRESUMO
The Suppressor of fused (Su(fu)) protein is known to be a negative regulator of Hedgehog (Hh) signal transduction in Drosophila imaginal discs and embryonic development. It is antagonized by the kinase Fused (Fu) since Su(fu) null mutations fully suppress the lack of Fu kinase activity. In this study, we overexpressed the Su(fu) gene in imaginal discs and observed opposing effects depending on the position of the cells, namely a repression of Hh target genes in cells receiving Hh and their ectopic expression in cells not receiving Hh. These effects were all enhanced in a fu mutant context and were suppressed by cubitus interruptus (Ci) overexpression. We also show that the Su(fu) protein is poly-phosphorylated during embryonic development and these phosphorylation events are altered in fu mutants. This study thus reveals an unexpected role for Su(fu) as an activator of Hh target gene expression in absence of Hh signal. Both negative and positive roles of Su(fu) are antagonized by Fused. Based on these results, we propose a model in which Su(fu) protein levels and isoforms are crucial for the modulation of the different Ci states that control Hh target gene expression.
Assuntos
Proteínas de Drosophila/metabolismo , Proteínas de Drosophila/fisiologia , Drosophila/metabolismo , Proteínas Repressoras/metabolismo , Transdução de Sinais , Animais , Proteínas de Ligação a DNA/metabolismo , Drosophila/embriologia , Proteínas de Drosophila/genética , Embrião não Mamífero/metabolismo , Olho/embriologia , Olho/metabolismo , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Proteínas Hedgehog , Masculino , Mutação , Fosforilação , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Processamento de Proteína Pós-Traducional , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Repressoras/genética , Fatores de Transcrição/metabolismo , Asas de Animais/embriologia , Asas de Animais/metabolismoRESUMO
BACKGROUND: In Drosophila and vertebrates, suppressor of fused (Su(fu)) proteins act as negative regulators of the Gli/Ci transcription factors, which mediate the transcriptional effects of Hh signalling. RESULTS: We sought for novel partners of Su(fu) in fly using the two-hybrid method. Most of the Su(fu) interactors thus identified are (or are likely to be) able to enter the nucleus. We focused on one of these putative partners, dMLF, which resembles vertebrate myelodysplasia/myeloid leukaemia factors 1 and 2. We demonstrate that dMLF binds specifically to Su(fu) in vitro and in vivo. Using a novel anti-dMLF antibody, we showed, that dMLF is a nuclear, chromosome-associated protein. We over-expressed a dMLF transgene in fly using an inducible expression system and showed that dMLF over-expression disrupts normal development, leading to either a lethal phenotype or adult structural defects associated with apoptosis and increased DNA synthesis. Furthermore, the dMLF-induced eye phenotype is enhanced by the loss of Su(fu) function, suggesting a genetic interaction between Su(fu) and dMLF. CONCLUSION: We propose that dSu(fu) and dMLF act together at the transcriptional level to coordinate patterning and proliferation during development.