The Hsp60C gene in the 25F cytogenetic region in Drosophila melanogaster is essential for tracheal development and fertility

Earlier studies have shown that of the four genes (Hsp60A, Hsp60B, Hsp60C, Hsp60D genes) predicted to encode the conserved Hsp60 family chaperones in Drosophila melanogaster, the Hsp60A gene (at the 10A polytene region) is expressed in all cell types of the organism and is essential from early embryonic stages, while the Hsp60B gene (at 21D region) is expressed only in testis, being essential for sperm individualization. In the present study, we characterized the Hsp60C gene (at 25F region), which shows high sequence homology with the other three Hsp60 genes of D. melanogaster. In situ hybridization of Hsp60C-specific riboprobe shows that expression of this gene begins in late embryonic stages (stage 14 onwards), particularly in the developing tracheal system and salivary glands; during larval and adult stages, it is widely expressed in many cell types but much more strongly in tracheae and in developing and differentiating germ cells. A P-insertion mutant (Hsp60C1 ) allele with the P transposon inserted at -251 position of the Hsp60C gene promoter was generated. This early larval recessive lethal mutation significantly reduces levels of Hsp60C transcripts in developing tracheae and this is associated with a variety of defects in the tracheal system, including lack of liquid clearance. About 10% of the homozygotes survive as weak, shortlived and completely sterile adults. Testes of the surviving mutant males are significantly smaller, with fewer spermatocytes, most of which do not develop beyond the round spermatid stage. In situ and Northern hybridizations show significantly reduced levels of the Hsp60C transcripts in Hsp60C1 homozygous adult males. The absence of early meiotic stages in the Hsp60C1 homozygous testes contrasts with the effect of testis-specific Hsp60B (21D) gene, whose mutation affects individualization of sperm bundles later in spermiogenesis. In view of the specific effects in tracheal development and in early stages of spermatogenesis, it is likely that, besides its functions as a chaperone, Hsp60C may have signalling functions and may also be involved in cation transport across the developing tracheal epithelial cells

The commonly used eye-specific sev-GAL4 and GMR-GAL4 drivers in Drosophila melanogaster are expressed in tissues other than eyes also

The binary GAL4-UAS system of conditional gene expression is widely used by Drosophila geneticists to target expression of the desired transgene in tissue of interest. In many studies, a preferred target tissue is the Drosophila eye, for which the sev-GAL4 and GMR-GAL4 drivers are most widely used since they are believed to be expressed exclusively in the developing eye cells. However, several reports have noted lethality following expression of certain transgenes under these GAL4 drivers notwithstanding the fact that eye is not essential for survival of the fly. Therefore, to explore the possibility that these drivers may also be active in tissues other than eye, we examined the expression of UAS-GFP reporter driven by the sev-GAL4 or GMR-GAL4 drivers. We found that both these drivers are indeed expressed in additional tissues, including a common set of specific neuronal cells in larval and pupal ventral and cerebral ganglia. Neither sev nor glass gene has so far been reported to be expressed in these neuronal cells. Expression pattern of sev-GAL4 driver parallels that of the endogenous Sevenless protein. In addition to cells in which sev-GAL4 is expressed, the GMR-GAL4 is expressed in several other larval cell types also. Further, two different GMR-GAL4 lines also show some specific differences in their expression domains outside the eye discs. These findings emphasize the need for a careful confirmation of the expression domains of a GAL4 driver being used in a given study, rather than relying only on the empirically claimed expression domains

Pleiotropic consequences of misexpression of the developmentally active and stress-inducible non-coding hsrω gene in Drosophila

The non-coding hsrω gene of Drosophila melanogaster is expressed in nearly all cell types and developmental stages. However, in the absence of conventional mutant alleles of this gene, its developmental functions remain largely unknown. In the present study, we used a variety of GAL4 drivers to overexpress or ablate this gene's transcripts in specific tissues and examined the developmental consequences thereof. Our results show that a balanced expression of these non-coding transcripts is critical for survival and normal development in all the tissue types tested, since any change in cellular levels of these transcripts in a given cell type generally has detrimental effects, with extreme cases resulting in organismal lethality, although in a few cases the misexpression of these transcripts also suppresses the mutant phenotype due to other genetic conditions. Evidence is also presented for existence of a new spliced variant of the hsrω-n nuclear transcript. Following the RNAi-mediated down-regulation of hsrω transcripts, the omega speckles disappear so that the nucleoplasmic hnRNPs get diffusely distributed, while upregulation of these transcripts results in greater sequestration of these proteins into omega speckle clusters; either of these conditions would affect activities of the hnRNPs and other hsrω-RNA interacting proteins, which is likely to have cascading consequences. The present findings, together with our earlier observations on effects of altered levels of the hsrω transcripts on induced apoptosis and expanded polyQ-mediated neurodegeneration, further confirm that ncRNA species like the hsrω, far from being evolutionary hangovers, provide critical information for important functions in normal cells

The hnRNP A1 homolog Hrp36 is essential for normal development, female fecundity, omega speckle formation and stress tolerance in Drosophila melanogaster

Hrp36/Hrb87F is one of the most abundant and well-characterized hnRNP A homolog in Drosophila and is shown to have roles in regulation of alternative splicing, heterochromatin formation, neurodegeneration, etc. Yet, hrp36 null individuals were reported to be viable and without any apparent phenotype, presumably because of overlapping functions provided by Hrp38 and related proteins. Here we show that loss of both copies of hrp36 gene slows down development with significant reduction in adult life span, decreased female fecundity and high sensitivity to starvation and thermal stresses. In the absence of Hrp36, the nucleoplasmic omega speckles are nearly completely disrupted. The levels of nuclear matrix protein Megator and the chromatin remodeller ISWI are significantly elevated in principal cells of larval Malpighian tubules, which also display additional endoreplication cycles and good polytene chromosomes. We suggest that besides the non-coding hsr omega-n transcripts, the Hrp36 protein is also a core constituent of omega speckles. The heat-shock-induced association of other hnRNPs at the hsr omega locus is affected in hrp36 null cells, which may be one of the reasons for their high sensitivity to cell stress. Therefore, in spite of the functional redundancy provided by Hrp38, Hrp36 is essential for normal development and for survival under conditions of stress

Heat shock genes-integrating cell survival and death

Heat shock induced gene expression and other cellular responses help limit the damage caused by stress and thus facilitate cellular recovery. Cellular damage also triggers apoptotic cell death through several pathways. This paper briefly reviews interactions of the major heat shock proteins with components of the apoptotic pathways. Hsp90, which acts as a chaperone for unstable signal transducers to keep them poised for activation, interacts with RIP and Akt and promotes NF-κB mediated inhibition of apoptosis; in addition it also blocks some steps in the apoptotic pathways. Hsp70 is mostly anti-apoptotic and acts at several levels like inhibition of translocation of Bax into mitochondria, release of cytochrome c from mitochondria, formation of apoptosome and inhibition of activation of initiator caspases. Hsp70 also modulates JNK, NF-κB and Akt signaling pathways in the apoptotic cascade. In contrast, Hsp60 has both anti- and pro-apoptotic roles. Cytosolic Hsp60 prevents translocation of the pro-apoptotic protein Bax into mitochondria and thus promotes cell survival but it also promotes maturation of procaspase-3, essential for caspase mediated cell death. Our recent in vivo studies show that RNAi for the Hsp60D in Drosophila melanogaster prevents induced apoptosis. Hsp27 exerts its anti-apoptotic influence by inhibiting cytochrome c and TNF-mediated cell death. αβ crystallin suppresses caspase-8 and cytochrome c mediated activation of caspase-3. Studies in our laboratory also reveal that absence or reduced levels of the developmentally active as well as stress induced non-coding hsrω transcripts, which are known to sequester diverse hnRNPs and related nuclear RNA-binding proteins, block induced apoptosis in Drosophila. Modulation of the apoptotic pathways by Hsps reflects their roles as "weak links" between various "hubs" in cellular networks. On the other hand, non-coding RNAs, by virtue of their potential to bind with multiple proteins, can act as "hubs" in these networks. In view of the integrative nature of living systems, it is not surprising that stress-induced genes, generally believed to primarily function in cell survival pathways, inhibit or even promote cell death pathways at multiple levels to ensure homeostasis at cell and/or organism level. The heat shock genes obviously do much more than merely help cells survive stress

Suppression of induced but not developmental apoptosis in drosophila by ayurvedic amalaki rasayana and rasa-sindoor

Earlier we showed formulation-specific beneficial effects of dietary supplement of Ayurvedic Amalaki Rasayana (AR, a herbal formulation) and Rasa-Sindoor (RS, a mercury-based organo-metallic formulation) on various biological parameters in Drosophila, parallel to traditional Ayurvedic literature. These formulations also suppressed cell death and pathology in fly models of neurodegeneration. To understand basis of inhibition of apoptosis, we examined effects of AR and RS on induced and developmental apoptosis in Drosophila. Dietary AR or RS significantly reduced apoptosis induced by GMR-GAL4-, sev-GAL4- or hs-GAL4-directed expression of Rpr, Hid or Grim (RHG) pro-apoptotic proteins or by GMR-GAL4-directed DIAP1-RNAi, resulting in significant restoration of organism's viability and eye morphology. AR or RS supplement enhanced levels of inhibitor of apoptosis proteins, DIAP1 and DIAP2, and of Bancal/Hrb57A, while the levels of RHG proteins and of initiator Dronc and effecter Drice caspases were reduced in non-apoptotic wild type as well as in RHG over-expressing tissues. Levels of Dronc or Drice remained unaffected in cells developmentally destined to die so that developmental apoptosis occurred normally. Elevated levels of DIAPs and reduced levels of RHG proteins and caspases reflect a more robust physiological state of AR or RS fed organisms allowing them to tolerate greater insults without triggering the cell-death response. Such homeostatic effects of these Rasayanas seem to contribute to ‘healthy ageing’, one of their effects suggested in traditional Ayurvedic practices

Ayurvedic Amalaki Rasayana and Rasa-Sindoor suppress neurodegeneration in fly models of Huntington’s and Alzheimer’s diseases

We examined two Ayurvedic Rasayana formulations, claimed to facilitate ‘healthy ageing’, for their role in neuroprotection in fly models of polyQ (127Q and Huntington’s) and Alzheimer’s disorders. Our earlier findings showed that dietary supplement of Amalaki Rasayana, a preparation derived from Indian gooseberry fruits, and Rasa-Sindoor, an organo-metallic Bhasma prepared from mercury and sulphur, improves general well-being of fruit flies. Here we show that dietary supplement of either of these formulations during larval period substantially suppressed neurodegeneration in fly models of polyQ and Alzheimer’s disorders without any side-effects. Dietary Amalaki Rasayana or Rasa-Sindoor prevented accumulation of inclusion bodies and heat shock proteins, suppressed apoptosis, elevated the levels of heterogeneous nuclear ribonucleoproteins and cAMP response element binding protein and at the same time improved the ubiquitin–proteasomal system for better protein clearance in affected cells. Our studies suggest, the potential of these Ayurvedic formulations in providing a holistic relief from the increasingly common neurodegenerative disorders

In vivo effects of traditional ayurvedic formulations in Drosophila melanogaster model relate with therapeutic applications

Background: Ayurveda represents the traditional medicine system of India. Since mechanistic details of therapy in terms of current biology are not available in Ayurvedic literature, modern scientific studies are necessary to understand its major concepts and procedures. It is necessary to examine effects of the whole Ayurvedic formulations rather than their “active” components as is done in most current studies. Methods: We tested two different categories of formulations, a Rasayana (Amalaki Rasayana or AR, an herbal derivative) and a Bhasma (Rasa-Sindoor or RS, an organo-metallic derivative of mercury), for effects on longevity, development, fecundity, stress-tolerance, and heterogeneous nuclear ribonucleoprotein (hnRNP) levels of Drosophila melanogaster using at least 200 larvae or flies for each assay. Results: A 0.5% (weight/volume) supplement of AR or RS affected life-history and other physiological traits in distinct ways. While the size of salivary glands, hnRNP levels in larval tissues, and thermotolerance of larvae/adult flies improved significantly following feeding either of the two formulations, the median life span and starvation resistance improved only with AR. Feeding on AR or RS supplemented food improved fecundity differently. Feeding of larvae and adults with AR increased the fecundity while the same with RS had opposite effect. On the contrary, feeding larvae on normal food and adults on AR supplement had no effect on fecundity but a comparable regime of feeding on RS-supplemented food improved fecundity. RS feeding did not cause heavy metal toxicity. Conclusions: The present study with two Ayurvedic formulations reveals formulation-specific effects on several parameters of the fly's life, which seem to generally agree with their recommended human usages in Ayurvedic practices. Thus, Drosophila, with its very rich genetic tools and well-worked-out developmental pathways promises to be a very good model for examining the cellular and molecular bases of the effects of different Ayurvedic formulations

The ISWI Chromatin Remodeler Organizes the hsrω ncRNA–Containing Omega Speckle Nuclear Compartments

The complexity in composition and function of the eukaryotic nucleus is achieved through its organization in specialized nuclear compartments. The Drosophila chromatin remodeling ATPase ISWI plays evolutionarily conserved roles in chromatin organization. Interestingly, ISWI genetically interacts with the hsrω gene, encoding multiple non-coding RNAs (ncRNA) essential, among other functions, for the assembly and organization of the omega speckles. The nucleoplasmic omega speckles play important functions in RNA metabolism, in normal and stressed cells, by regulating availability of hnRNPs and some other RNA processing proteins. Chromatin remodelers, as well as nuclear speckles and their associated ncRNAs, are emerging as important components of gene regulatory networks, although their functional connections have remained poorly defined. Here we provide multiple lines of evidence showing that the hsrω ncRNA interacts in vivo and in vitro with ISWI, regulating its ATPase activity. Remarkably, we found that the organization of nucleoplasmic omega speckles depends on ISWI function. Our findings highlight a novel role for chromatin remodelers in organization of nucleoplasmic compartments, providing the first example of interaction between an ATP-dependent chromatin remodeler and a large ncRNA

Human sat III and Drosophila hsrω transcripts: a common paradigm for regulation of nuclear RNA processing in stressed cells

Exposure of cells to stressful conditions elicits a highly conserved defense mechanism termed the heat shock response, resulting in the production of specialized proteins which protect the cells against the deleterious effects of stress. The heat shock response involves not only a widespread inhibition of the ongoing transcription and activation of heat shock genes, but also important changes in post-transcriptional processing. In particular, a blockade in splicing and other post-transcriptional processing has been described following stress in different organisms, together with an altered spatial distribution of the proteins involved in these activities. However, the specific mechanisms that regulate these activities under conditions of stress are little understood. Non-coding RNA molecules are increasingly known to be involved in the regulation of various activities in the cell, ranging from chromatin structure to splicing and RNA degradation. In this review, we consider two non-coding RNAs, the hsrω transcripts in Drosophila and the sat III transcripts in human cells, that seem to be involved in the dynamics of RNA-processing factors in normal and/or stressed cells, and thus provide new paradigms for understanding transcriptional and post-transcriptional regulations in normal and stressed cells