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

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

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

The Developmentally Active and Stress-Inducible Noncoding hsrω Gene Is a Novel Regulator of Apoptosis in Drosophila

The large nucleus limited noncoding hsrω-n RNA of Drosophila melanogaster is known to associate with a variety of heterogeneous nuclear RNA-binding proteins (hnRNPs) and certain other RNA-binding proteins to assemble the nucleoplasmic omega speckles. In this article, we show that RNAi-mediated depletion of this noncoding RNA dominantly suppresses apoptosis, in eye and other imaginal discs, triggered by induced expression of Rpr, Grim, or caspases (initiator as well as effector), all of which are key regulators/effectors of the canonical caspase-mediated cell death pathway. We also show, for the first time, a genetic interaction between the noncoding hsrω transcripts and the c-Jun N-terminal kinase (JNK) signaling pathway since downregulation of hsrω transcripts suppressed JNK activation. In addition, hsrω-RNAi also augmented the levels of Drosophila Inhibitor of Apoptosis Protein 1 (DIAP1) when apoptosis was activated. Suppression of induced cell death following depletion of hsrω transcripts was abrogated when the DIAP1-RNAi transgene was coexpressed. Our results suggest that the hsrω transcripts regulate cellular levels of DIAP1 via the hnRNP Hrb57A, which physically interacts with DIAP1, and any alteration in levels of the hsrω transcripts in eye disc cells enhances association between these two proteins. Our studies thus reveal a novel regulatory role of the hsrω noncoding RNA on the apoptotic cell death cascade through multiple paths. These observations add to the diversity of regulatory functions that the large noncoding RNAs carry out in the cells' life

Improved Activities of CREB Binding Protein, Heterogeneous Nuclear Ribonucleoproteins and Proteasome Following Downregulation of Noncoding hsrω Transcripts Help Suppress Poly(Q) Pathogenesis in Fly Models

Following earlier reports on modulation of poly(Q) toxicity in Drosophila by the developmentally active and stress-inducible noncoding hsrω gene, we investigated possible mediators of this modulation. RNAi-mediated downregulation of the large nuclear hsrω-n transcript, which organizes the nucleoplasmic omega speckles, suppressed the enhancement of poly(Q) toxicity brought about by reduced availability of the heterogeneous nuclear ribonucleoprotein (hnRNP) Hrb87F and of the transcriptional regulator, cAMP response element binding (CREB) binding protein (CBP). Levels of CBP RNA and protein were reciprocally affected by hsrω transcript levels in eye disc cells. Our data suggest that CBP and hnRNPs like Hrb57A and Hrb87F physically interact with each other. In addition, downregulation of hsrω transcripts partially rescued eye damage following compromised proteasome activity, while overexpression of hsrω and/or poly(Q) proteins disrupted the proteasomal activity. Rescue of poly(Q) toxicity by hsrω-RNAi required normal proteasomal function. We suggest that hsrω-RNAi suppresses poly(Q) toxicity by elevating cellular levels of CBP, by enhancing proteasome-mediated clearance of the pathogenic poly(Q) aggregates, and by inhibiting induced apoptosis. The direct and indirect interactions of the hsrω transcripts with a variety of regulatory proteins like hnRNPs, CBP, proteasome, Drosophila inhibitor of apoptosis protein 1 (DIAP1), etc., reinforce the view that the noncoding hsrω RNA functions as a “hub” in cellular networks to maintain homeostasis by coordinating the functional availability of crucial cellular regulatory proteins

RNAi for the large non-coding hsrω transcripts suppresses polyglutamine pathogenesis in Drosophila models

Polyglutamine diseases are a class of inherited neurodegenerative disorders, characterized by expansion of CAG trinucleotide repeats translated into elongated glutamine tracts within the mutant proteins. Overexpression of the non-coding hsrω transcripts has been shown to dominantly enhance polyQ induced cytotoxicity in Drosophila. In the present study we demonstrate that RNA interference mediated downregulation of hsrω-n transcripts is sufficient to suppress pathogenesis in several Drosophila models of human polyQ neurodegenerative diseases. Loss of hsrω-n RNA not only suppresses the eye-specific degeneration mediated by GMR-GAL4 driven expression of the 127Q or MJDtr-Q78 or ataxin1 82Q or httex1p Q93 transgene, but also rescues premature death of flies expressing the expanded polyQ proteins pan-neuronally using the elav-GAL4 driver. We further demonstrate that the morphological and functional rescue of polyQ toxicity observed upon hsrω-n RNAi is associated with substantial reduction of polyQ protein aggregation without affecting transcription of the 127Q transgene. Unlike in the polyQ expressing cells, co-expression of hsrω-n RNAi also abolishes the induction of Hsp70. These results suggest that the hsrω transcripts have a role in early stages of polyQ aggregate formation. Interestingly, hsrω-RNAi has, at best, only a marginal effect on neuropathy following overexpression of normal or mutant tau protein in flies. Functional analogues of the large non-coding hsrω transcripts in human thus appear to be promising candidates as therapeutic targets for the polyQ-mediated neurodegenerative diseases

The hsr&#969;<SUP>05241</SUP> allele of the noncoding hsr&#969; gene of Drosophila melanogaster is not responsible for male sterility as reported earlier

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The large noncoding hsrω-n transcripts are essential for thermotolerance and remobilization of hnRNPs, HP1 and RNA polymerase II during recovery from heat shock in Drosophila

The hs-GAL4t -driven expression of the hsrω-RNAi transgene or EP93D allele of the noncoding hsrω resulted in global down- or upregulation, respectively, of the large hsrω-n transcripts following heat shock. Subsequent to temperature shock, hsrω-null or those expressing hsrω-RNAi or the EP93D allele displayed delayed lethality of most embryos, first or third instar larvae. Three-day-old hsrω-null flies mostly died immediately or within a day after heat shock. Heat-shock-induced RNAi or EP expression in flies caused only a marginal lethality but severely affected oogenesis. EP allele or hsrω-RNAi expression after heat shock did not affect heat shock puffs and Hsp70 synthesis. Both down- and upregulation of hsrω-n transcripts suppressed reappearance of the hsrω-n transcript-dependent nucleoplasmic omega speckles during recovery from heat shock. Hrp36, heterochromatin protein 1, and active RNA pol II in unstressed or heat-shocked wild-type or hsrω-null larvae or those expressing the hs-GAL4t -driven hsrω-RNAi or the EP93D allele were comparably distributed on polytene chromosomes. Redistribution of these proteins to pre-stress locations after a 1- or 2-h recovery was severely compromised in glands with down- or upregulated levels of hsrω-n transcripts after heat shock. The hsrω-null unstressed cells always lacked omega speckles and little Hrp36 moved to any chromosome region following heat shock, and its relocation to chromosome regions during recovery was also incomplete. This present study reveals for the first time that the spatial restoration of key regulatory factors like hnRNPs, HP1, or RNA pol II to their pre-stress nuclear targets in cells recovering from thermal stress is dependent upon critical level of the large hsrω-n noncoding RNA. In the absence of their relocation to pre-stress chromosome sites, normal developmental gene activity fails to be restored, which finally results in delayed organismal death

tRNA overexpression rescues peripheral neuropathy caused by mutations in tRNA synthetase

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