ABSTRACT
Nanostructures of pores and protrusions in the insect cuticle modify molecular permeability and surface wetting and help insects sense various environmental cues. However, the cellular mechanisms that modify cuticle nanostructures are poorly understood. Here, we elucidate how insect-specific Osiris family genes are expressed in various cuticle-secreting cells in the Drosophila head during the early stages of cuticle secretion and cover nearly the entire surface of the head epidermis. Furthermore, we demonstrate how each sense organ cell with various cuticular nanostructures expressed a unique combination of Osiris genes. Osiris gene mutations cause various cuticle defects in the corneal nipples and pores of the chemosensory sensilla. Thus, our study emphasizes on the importance of Osiris genes for elucidating cuticle nanopatterning in insects.
Subject(s)
Drosophila Proteins , Animals , Drosophila Proteins/genetics , Drosophila Proteins/metabolism , Drosophila melanogaster/genetics , Sensilla/metabolism , Multigene Family , Mutation , Nanostructures/chemistry , Drosophila/geneticsABSTRACT
Nanometer-level patterned surface structures form the basis of biological functions, including superhydrophobicity, structural coloration, and light absorption [1-3]. In insects, the cuticle overlying the olfactory sensilla has multiple small (50- to 200-nm diameter) pores [4-8], which are supposed to function as a filter that admits odorant molecules, while preventing the entry of larger airborne particles and limiting water loss. However, the cellular processes underlying the patterning of extracellular matrices into functional nano-structures remain unknown. Here, we show that cuticular nanopores in Drosophila olfactory sensilla originate from a curved ultrathin film that is formed in the outermost envelope layer of the cuticle and secreted from specialized protrusions in the plasma membrane of the hair forming (trichogen) cell. The envelope curvature coincides with plasma membrane undulations associated with endocytic structures. The gore-tex/Osiris23 gene encodes an endosomal protein that is essential for envelope curvature, nanopore formation, and odor receptivity and is expressed specifically in developing olfactory trichogen cells. The 24-member Osiris gene family is expressed in cuticle-secreting cells and is found only in insect genomes. These results reveal an essential requirement for nanopores for odor reception and identify Osiris genes as a platform for investigating the evolution of surface nano-fabrication in insects.
Subject(s)
Drosophila melanogaster/ultrastructure , Sensilla/ultrastructure , Animals , Female , Microscopy, Electron, Transmission , Nanopores/ultrastructureABSTRACT
To compensate for accumulating damages and cell death, adult homeostasis (e.g., body fluids and secretion) requires organ regeneration, operated by long-lived stem cells. How stem cells can survive throughout the animal life remains poorly understood. Here we show that the transcription factor Shavenbaby (Svb, OvoL in vertebrates) is expressed in renal/nephric stem cells (RNSCs) of Drosophila and required for their maintenance during adulthood. As recently shown in embryos, Svb function in adult RNSCs further needs a post-translational processing mediated by the Polished rice (Pri) smORF peptides and impairing Svb function leads to RNSC apoptosis. We show that Svb interacts both genetically and physically with Yorkie (YAP/TAZ in vertebrates), a nuclear effector of the Hippo pathway, to activate the expression of the inhibitor of apoptosis DIAP1. These data therefore identify Svb as a nuclear effector in the Hippo pathway, critical for the survival of adult somatic stem cells.
Subject(s)
Adult Stem Cells/metabolism , DNA-Binding Proteins/metabolism , Drosophila Proteins/metabolism , Intracellular Signaling Peptides and Proteins/metabolism , Nuclear Proteins/metabolism , Protein Serine-Threonine Kinases/metabolism , Trans-Activators/metabolism , Transcription Factors/metabolism , Animals , Apoptosis/genetics , Apoptosis/physiology , DNA-Binding Proteins/genetics , Drosophila , Drosophila Proteins/genetics , In Situ Nick-End Labeling , Intracellular Signaling Peptides and Proteins/genetics , Nuclear Proteins/genetics , Protein Serine-Threonine Kinases/genetics , Signal Transduction/genetics , Signal Transduction/physiology , Trans-Activators/genetics , Transcription Factors/genetics , YAP-Signaling ProteinsABSTRACT
The outer surface of insects is covered by the cuticle, which is derived from the apical extracellular matrix (aECM). The aECM is secreted by epidermal cells during embryogenesis. The aECM exhibits large variations in structure, function, and constituent molecules, reflecting the enormous diversity in insect appearances. To investigate the molecular principles of aECM organization and function, here we studied the role of a conserved aECM protein, the ZP domain protein Trynity, in Drosophila melanogaster. We first identified trynity as an essential gene for epidermal barrier function. trynity mutation caused disintegration of the outermost envelope layer of the cuticle, resulting in small-molecule leakage and in growth and molting defects. In addition, the tracheal tubules of trynity mutants showed defects in pore-like structures of the cuticle, and the mutant tracheal cells failed to absorb luminal proteins and liquid. Our findings indicated that trynity plays essential roles in organizing nano-level structures in the envelope layer of the cuticle that both restrict molecular trafficking through the epidermis and promote the massive absorption pulse in the trachea.
Subject(s)
Drosophila Proteins/metabolism , Drosophila/metabolism , Extracellular Matrix/metabolism , Animals , Behavior, Animal/physiology , CRISPR-Cas Systems/genetics , Drosophila/growth & development , Drosophila Proteins/genetics , Embryo, Nonmammalian/metabolism , Epidermis/metabolism , Epidermis/pathology , Gene Knockout Techniques , Larva/growth & development , Larva/metabolism , Osmolar Concentration , Trachea/metabolismABSTRACT
Animal development fundamentally relies on the precise control, in space and time, of genome expression. Whereas we have a wealth of information about spatial patterning, the mechanisms underlying temporal control remain poorly understood. Here we show that Pri peptides, encoded by small open reading frames, are direct mediators of the steroid hormone ecdysone for the timing of developmental programs in Drosophila. We identify a previously uncharacterized enzyme of ecdysone biosynthesis, GstE14, and find that ecdysone triggers pri expression to define the onset of epidermal trichome development, through post-translational control of the Shavenbaby transcription factor. We show that manipulating pri expression is sufficient to either put on hold or induce premature differentiation of trichomes. Furthermore, we find that ecdysone-dependent regulation of pri is not restricted to epidermis and occurs over various tissues and times. Together, these findings provide a molecular framework to explain how systemic hormonal control coordinates specific programs of differentiation with developmental timing.
Subject(s)
Arrestins/metabolism , Drosophila Proteins/metabolism , Drosophila melanogaster/metabolism , Ecdysone/metabolism , Gene Expression Regulation, Developmental/physiology , Glutathione Transferase/metabolism , Receptors, Steroid/metabolism , Animals , Arrestins/genetics , Cell Differentiation/genetics , Drosophila Proteins/genetics , Ecdysone/genetics , Glutathione Transferase/genetics , Mutation/genetics , Receptors, Steroid/genetics , Signal Transduction/physiology , Transaldolase/genetics , Transaldolase/metabolismABSTRACT
In Drosophila, the PIWI proteins, Aubergine (Aub), AGO3, and Piwi are expressed in germlines and function in silencing transposons by associating with PIWI-interacting RNAs (piRNAs). Recent studies show that PIWI proteins contain symmetric dimethyl-arginines (sDMAs) and that dPRMT5/Capsuleen/DART5 is the modifying enzyme. Here, we show that Tudor (Tud), one of Tud domain-containing proteins, associates with Aub and AGO3, specifically through their sDMA modifications and that these three proteins form heteromeric complexes. piRNA precursor-like molecules are detected in these complexes. The expression levels of Aub and AGO3, along with their degree of sDMA modification, were not changed by tud mutations. However, the population of transposon-derived piRNAs associated with Aub and AGO3 was altered by tud mutations, whereas the total amounts of small RNAs on Aub and AGO3 was increased. Loss of dprmt5 did not change the stability of Aub, but impaired its association with Tud and lowered piRNA association with Aub. Thus, in germline cells, piRNAs are quality-controlled by dPRMT5 that modifies PIWI proteins, in tight association with Tud.
Subject(s)
Drosophila Proteins/metabolism , Drosophila melanogaster/metabolism , Membrane Transport Proteins/metabolism , Protein Methyltransferases/metabolism , Amino Acid Sequence , Animals , Arginine/analogs & derivatives , Arginine/chemistry , Chromatography, Liquid/methods , Databases, Protein , Gene Expression Regulation , Mass Spectrometry/methods , Molecular Sequence Data , Mutation , Protein-Arginine N-Methyltransferases , RNA Interference , Sequence Homology, Amino AcidABSTRACT
PIWI-interacting RNAs (piRNAs) silence retrotransposons in Drosophila germ lines by associating with the PIWI proteins Argonaute 3 (AGO3), Aubergine (Aub) and Piwi. piRNAs in Drosophila are produced from intergenic repetitive genes and piRNA clusters by two systems: the primary processing pathway and the amplification loop. The amplification loop occurs in a Dicer-independent, PIWI-Slicer-dependent manner. However, primary piRNA processing remains elusive. Here we analysed piRNA processing in a Drosophila ovarian somatic cell line where Piwi, but not Aub or AGO3, is expressed; thus, only the primary piRNAs exist. In addition to flamenco, a Piwi-specific piRNA cluster, traffic jam (tj), a large Maf gene, was determined as a new piRNA cluster. piRNAs arising from tj correspond to the untranslated regions of tj messenger RNA and are sense-oriented. piRNA loading on to Piwi may occur in the cytoplasm. zucchini, a gene encoding a putative cytoplasmic nuclease, is required for tj-derived piRNA production. In tj and piwi mutant ovaries, somatic cells fail to intermingle with germ cells and Fasciclin III is overexpressed. Loss of tj abolishes Piwi expression in gonadal somatic cells. Thus, in gonadal somatic cells, tj gives rise simultaneously to two different molecules: the TJ protein, which activates Piwi expression, and piRNAs, which define the Piwi targets for silencing.
Subject(s)
Drosophila Proteins/metabolism , Drosophila melanogaster/metabolism , Maf Transcription Factors, Large/metabolism , Proto-Oncogene Proteins/metabolism , RNA-Induced Silencing Complex/metabolism , RNA/metabolism , Animals , Argonaute Proteins , Cell Adhesion Molecules, Neuronal/metabolism , Cell Line , Drosophila Proteins/genetics , Drosophila melanogaster/genetics , Endoribonucleases/metabolism , Female , Genes, Insect/genetics , Genetic Loci/genetics , Maf Transcription Factors, Large/genetics , Male , Ovary/cytology , Ovary/metabolism , Phenotype , Proto-Oncogene Proteins/genetics , RNA/biosynthesis , RNA/genetics , RNA Interference , RNA Processing, Post-Transcriptional , RNA-Induced Silencing Complex/genetics , Testis/cytology , Testis/metabolismABSTRACT
Genetic studies have shown that Aubergine (Aub), one of the Piwi subfamily of Argonautes in Drosophila, is essential for germ cell formation and maintaining fertility. aub mutations lead to the accumulation of retrotransposons in ovaries and testes, and Stellate transcripts in testes. Aub in ovaries associates with a variety of Piwi-interacting RNAs (piRNAs) derived from repetitive intergenic elements including retrotransposons. Here we found that Aub in testes also associates with various kinds of piRNAs. Although in ovaries Aub-associated piRNA populations are quite diverse, piRNAs with Aub in testes show a strong bias. The most abundant piRNAs were those corresponding to antisense transcripts of Suppressor of Stellate [Su(Ste)] genes known to be involved in Stellate gene silencing. The second most abundant class was made up of those from chromosome X and showed strong complementarity to vasa transcripts. Immunopurified Aub-piRNA complexes from testes displayed activity in cleaving target RNA containing sequences complementary to Stellate and vasa transcripts. These results provide the first biochemical insights into gene silencing mechanisms mediated by Aub and piRNAs in fly testes.
Subject(s)
Drosophila Proteins/metabolism , Drosophila/genetics , Gonads/metabolism , Peptide Initiation Factors/metabolism , RNA Interference , RNA, Small Interfering/metabolism , Animals , Base Sequence , Drosophila/metabolism , Drosophila Proteins/genetics , Female , Fluorescent Antibody Technique , Germ Cells/metabolism , Male , Models, Genetic , Molecular Sequence Data , Repressor Proteins/genetics , Repressor Proteins/metabolism , Testis/metabolismABSTRACT
Transcriptome analyses in eukaryotes, including mice and humans, have identified polyA-containing transcripts that lack long open reading frames (ORFs; >100 amino acids). These transcripts are believed most likely to function as non-coding RNAs, but their translational capacities and biological activities have not been characterized in detail. Here, we report that polished rice (pri), which was previously identified as a gene for a non-coding RNA in Drosophila, is in fact transcribed into a polycistronic mRNA that contains evolutionarily conserved short ORFs that encode 11 or 32 amino acid-long peptides. pri was expressed in all epithelial tissues during embryogenesis. The loss of pri function completely eliminated apical cuticular structures, including the epidermal denticles and tracheal taenidia, and also caused defective tracheal-tube expansion. We found that pri is essential for the formation of specific F-actin bundles that prefigures the formation of the denticles and taenidium. We provide evidences that pri acts non-cell autonomously and that four of the conserved pri ORFs are functionally redundant. These results demonstrate that pri has essential roles in epithelial morphogenesis by regulating F-actin organization.
Subject(s)
Actins/metabolism , Drosophila/embryology , Embryonic Development/genetics , Epithelium/embryology , Peptides/metabolism , RNA, Messenger/genetics , Animals , Base Sequence , Cell Differentiation/genetics , Conserved Sequence/genetics , Drosophila/cytology , Drosophila/metabolism , Epithelium/metabolism , Evolution, Molecular , Gene Expression Regulation, Developmental/genetics , Genes/genetics , Molecular Sequence Data , Open Reading Frames/genetics , Peptides/genetics , Sequence Homology, Nucleic AcidABSTRACT
RNAi is a gene-silencing phenomenon mediated by double-stranded RNA (dsRNA) and has become a powerful tool to elucidate gene function. To accomplish rapid construction of transgenes expressing dsRNA in Drosophila, we developed a novel transformation vector, pRISE, which contains an inverted repeat of the attR1-ccdB-attR2 cassette for in vitro recombination and a pentameric GAL4 binding site for conditional expression. These features enabled us to construct RNAi transgenes without a complicated cloning scheme. In cultured cells and transgenic flies, pRISE constructs carrying dsRNA transgenes induced effective RNAi against an EGFP transgene and the endogenous white gene, respectively. These results indicate that pRISE is a convenient transformation vector for studies of multiple Drosophila genes for which functional information is lacking.
Subject(s)
DNA Transposable Elements/genetics , Drosophila/genetics , Genetic Vectors/chemical synthesis , RNA, Small Interfering/genetics , Transgenes , ATP-Binding Cassette Transporters/metabolism , Animals , Animals, Genetically Modified , Cells, Cultured , Drosophila Proteins/deficiency , Drosophila Proteins/metabolism , Eye Proteins/metabolism , RNA Interference , Recombination, Genetic , Transformation, GeneticABSTRACT
One of the most surprising results to emerge from mammalian cDNA sequencing projects is that thousands of mRNA-like non-coding RNAs (ncRNAs) are expressed and constitute at least 10% of poly(A)(+) RNAs. In most cases, however, the functions of these RNA molecules remain unclear. To clarify the biological significance of mRNA-like ncRNAs, we computationally screened 11,691 Drosophila melanogaster full-length cDNAs. After eliminating presumable protein-coding transcripts, 136 were identified as strong candidates for mRNA-like ncRNAs. Although most of these putative ncRNAs are found throughout the Drosophila genus, predicted amino acid sequences are not conserved even in related species, suggesting that these transcripts are actually non-coding RNAs. In situ hybridization analyses revealed that 35 of the transcripts are expressed during embryogenesis, of which 27 were detected only in specific tissues including the tracheal system, midgut primordial cells, visceral mesoderm, germ cells and the central and peripheral nervous system. These highly regulated expression patterns suggest that many mRNA-like ncRNAs play important roles in multiple steps of organogenesis and cell differentiation in Drosophila. This is the first report that the majority of mRNA-like ncRNAs in a model organism are expressed in specific tissues and cell types.
Subject(s)
Drosophila/genetics , RNA, Messenger/genetics , RNA, Untranslated/genetics , Amino Acid Sequence , Animals , Base Sequence , Cell Differentiation/genetics , Conserved Sequence , DNA, Complementary/analysis , DNA, Complementary/genetics , Drosophila/embryology , Embryonic Development/genetics , Evolution, Molecular , Gene Expression Regulation, Developmental , Models, Genetic , Open Reading Frames/genetics , Organogenesis/genetics , RNA, Messenger/chemistry , RNA, Untranslated/chemistry , Species Specificity , Transcription, GeneticABSTRACT
BACKGROUND: Bacterial tmRNA (10Sa RNA) is involved in a trans-translation reaction, which contributes to the degradation of incompletely synthesized peptides and the recycling of stalled ribosomes. To investigate the physiological roles of this reaction in Bacillus subtilis, we devised a system for detecting the proteins that are subject to in vivo trans-translation. RESULTS: The wild-type tmRNA gene (ssrA) in the genome was replaced by a variant ssrA encoding a tag-peptide sequence containing six histidine residues (His-tag) and two aspartic acids at the C-terminus. The His-tagged proteins that accumulated in the cells without degradation were fractionated by Ni2+-NTA column and gel electrophoresis and were detected by Western blotting with an anti-His-tag antibody. The results showed that the trans-translation occurred more frequently at a high temperature (50 degrees C) than at a low temperature (37 degrees C). Two-dimensional (2D) gel electrophoresis of the products revealed many distinct spots, which represent specific target proteins for the trans-translation reaction. Furthermore, the 2D gel patterns of the products from cells cultured at high and low temperatures were apparently different. Several tagged proteins were identified by the N-terminal amino acid sequences of the products. CONCLUSION: Trans-translation occurs more frequently at high temperature than at low temperature, and different proteins are tagged at different temperatures.