The hs-GAL4(t)-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-GAL4(t)-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.

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.

We exploited the high Ras activity induced differentiation of supernumerary R7 cells in Drosophila eyes to examine if hsrω lncRNAs influence active Ras signaling. Surprisingly, either down-or up-regulation of hsrω lncRNAs in sev-GAL4>Ras V12 expressing eye discs resulted in complete pupal lethality and substantially greater increase in R7 photoreceptor number at the expense of cone cells. Enhanced nuclear p-MAPK and presence of sev-GAL4 driven Ras V12 bound RafRBDFLAG in cells not expressing the sev-GAL4 driver indicated non-cell autonomous spread of Ras signaling when hsrω levels were co-altered. RNAsequencing revealed that down-and up-regulation of hsrω transcripts in sev-GAL4>Ras V12 expressing eye discs elevated transcripts of positive or negative modulators, respectively, of Ras signaling so that either condition enhances it. Altered hsrω transcript levels in sev-GAL4>Ras V12 expressing discs also affected sn/sno/sca RNAs and some other RNA processing transcript levels. Post-transcriptional changes due to the disrupted intra-cellular dynamicity of omega speckle associated hnRNPs and other RNA-binding proteins that follow down-or up-regulation of hsrω lncRNAs appear to be responsible for the further elevated Ras signaling. Cell autonomous and non-autonomous enhancement of Ras signaling by lncRNAs like hsrω has implications for cell signaling during high Ras activity commonly associated with some cancers. HighlightsOur findings highlight roles of hsrω lncRNAs in conditionally modulating the important Ras signaling pathway and provide evidence for cell non-autonomous Ras signaling in Drosophila eye discs..

To examine role of hsrω nuclear lncRNAs in Ras signaling cascade, we down-or up-regulated these transcripts in eye discs of Drosophila expressing sev-GAL4 driven activated ras V12 transgene. The sev-GAL4 driven Ras V12 transgene expression dependent late pupal lethality and extra R7 photoreceptors in ommatidia, were significantly enhanced when levels of hsrω lncRNA were down/up-regulated. This was associated with enhanced p-MAPK expression, reduced Yan levels, and greater association of RafRBDFLAG with Ras, indicating elevated Ras activation which was both cell autonomous and non-autonomous. RNAseq analysis revealed significant increase in expression of certain sno/sn/scaRNAs and some RNA processing genes in sevGAL4>Ras V12 which was further modulated when hsrωRNA levels were co-altered. Downregulation of hsrωRNAs elevated positive modulators of Ras signaling while their up-regulation reduced expression of negative modulators of Ras signaling, and thus both conditions have similar outcome. Further enhancement of activity of hyperactive Ras following changes in hsrω lncRNA levels in cell autonomous as well as non-autonomous manner emphasizes roles of lncRNAs in cell signaling during development and disease conditions associated with hyperactive Ras pathway mutants. SummaryOur findings highlight roles of hsrω lncRNA in conditionally modulating the important Ras signaling pathway and provide evidence for cell non-autonomous Ras signaling in Drosophila eye discs.

We examined interactions between Hsp83 and hsrω lncRNAs in hsrω 66 Hsp90GFP homozygotes, which almost completely lack hsrω lncRNAs but over-express Hsp83. All +/+; hsrω 66 Hsp90GFP progeny died before third instar. Rare Sp/CyO; hsrω 66 Hsp90GFP reached third instar stage but phenocopied l(2)gl mutants, dying after prolonged larval life, becoming progressively bulbous and transparent with enlarged brain. Additionally, ventral ganglia were elongated. However, hsrω 66 Hsp90GFP/TM6B heterozygotes, carrying +/+ or Sp/CyO second chromosomes, developed normally. Total RNA sequencing (+/+, +/+; hsrω 66 /hsrω 66 , Sp/CyO; hsrω 66 /hsrω 66 , +/+; Hsp90GFP/Hsp90GFP, and Sp/CyO; hsrω 66 Hsp90GFP/hsrω 66 Hsp90GFP late third instar larvae) revealed similar effects on many genes in hsrω 66 and Hsp90GFP homozygotes. Besides additive effect on many of them, numerous additional genes were affected in Sp/CyO; hsrω 66 Hsp90GFP larvae, with l(2)gl and several genes regulating CNS being highly down-regulated in surviving Sp/CyO; hsrω 66 Hsp90GFP larvae, but not in hsrω 66 or Hsp90GFP single mutants. Hsp83 binds at these gene promoters. Several omega speckle associated hnRNPs too may bind with these genes and transcripts. Hsp83-hnRNP interactions are also known. Thus, elevated Hsp83 in altered hnRNP distribution and dynamics, following absence of hsrω lncRNAs and omega speckles, background can severely perturb regulatory circuits with unexpected consequences, including down-regulation of tumor suppressor gene like l(2)gl.

One sentence AbstractElevated JNK activity following predominant over-expression of activated Ras or Reaper or Tak1 in Drosophila larval eye discs does not delay pupation but reduces post-pupariation ecdysone pulse via elevated Dilp8 and reduced Ptth, which results in early pupal death. AbstractWe examined reasons for early pupal death following expression of certain transgenes with predominantly eye-disc specific GMR-GAL4 or sev-GAL4 drivers in Drosophila. The GMR-GAL4 or sev-GAL4 driven expression of UAS-Ras1 V12 transgene, producing activated Ras, resulted in early (~25-30Hr after pupa formation, APF) and late pupal death, respectively. Coexpression of UAS-hsrω-RNAi transgene or EP3037 to down or up-regulate the hsrω lncRNAs with sev-GAL4>UAS-Ras1 V12 advanced the death to 25-30Hr APF. The normal 8-12Hr APF ecdysone surge was absent in the early dying pupae. Exogenous ecdysone provided between 8-20Hr APF partially suppressed their early death. Microarray, qRT-PCR and immunostaining revealed substantial increase in some JNK pathway members in late larval eye discs, and of Dilp8 in eye discs, reduced transcripts of ptth in brain lobes and of ecdysone biosynthesis enzymes in prothoracic glands of 8-9Hr old pupae in genotypes that show early pupal death. We propose that the high JNK activity in eye discs of GMR-GAL4>UAS-Ras1 V12 and sev-GAL4>UAS-Ras1 V12 with altered hsrω transcript levels triggers greater Dilp8 secretion from them soon after pupa formation, which inhibits post-pupal ecdysone synthesis in prothoracic gland, leading to early pupal death. The early pupal death following GMR-GAL4 driven expression of UAS-rpr or UAS-tak1 was also associated with comparable enhanced JNK and Dilp8 signaling in eye discs. This study highlights how mis-regulated signaling in one tissue can have global deleterious consequences through downstream events in other tissues, reemphasizing role of inter organ signaling in life of an organism.

Background Dilp8‐mediated inhibition of ecdysone synthesis and pupation in holometabolous insects maintains developmental homeostasis through stringent control of timing and strength of molting signals. We examined reasons for normal pupation but early pupal death observed in certain cases. Results Overexpression of activated Ras in developing eye/wing discs inhibited Ptth expression in brain via upregulated JNK signaling mediated Dilp8 secretion from imaginal discs, which inhibited ecdysone synthesis in prothoracic gland after pupariation, leading to death of ~25‐ to 30‐hour‐old pupae. Inhibition of elevated Ras signaling completely rescued early pupal death while post‐pupation administration of ecdysone to organisms with elevated Ras signaling in eye discs partially rescued their early pupal death. Unlike the earlier known Dilp8 action in delaying pupation, hyperactivated Ras mediated elevation of pJNK signaling in imaginal discs caused Dilp8 secretion after pupariation. Ectopic expression of certain other transgene causing pupal lethality similarly enhanced pJNK and early pupal Dilp8 levels. Suboptimal ecdysone levels after 8 hours of pupation prevented the early pupal metamorphic changes and caused organismal death. Conclusions Our results reveal early pupal stage as a novel Dilp8 mediated post‐pupariation checkpoint and provide further evidence for interorgan signaling during development, wherein a peripheral tissue influences the CNS driven endocrine function.

We examined interactions between Hsp83 and hsrω lncRNAs in hsrω 66 Hsp90GFP homozygotes, which almost completely lack hsrω lncRNAs but over-express Hsp83. All +/+; hsrω 66 Hsp90GFP progeny died before third instar. Rare Sp/CyO; hsrω 66 Hsp90GFP reached third instar stage but phenocopied l(2)gl mutants, dying after prolonged larval life, becoming progressively bulbous and transparent with enlarged brain. Additionally, ventral ganglia were elongated. However, hsrω 66 Hsp90GFP/TM6B heterozygotes, carrying +/+ or Sp/CyO second chromosomes, developed normally. Total RNA sequencing (+/+, +/+; hsrω 66 /hsrω 66 , Sp/CyO; hsrω 66 /hsrω 66 , +/+; Hsp90GFP/Hsp90GFP, and Sp/CyO; hsrω 66 Hsp90GFP/hsrω 66 Hsp90GFP late third instar larvae) revealed similar effects on many genes in hsrω 66 and Hsp90GFP homozygotes. Besides additive effect on many of them, numerous additional genes were affected in Sp/CyO; hsrω 66 Hsp90GFP larvae, with l(2)gl and several genes regulating CNS being highly down-regulated in surviving Sp/CyO; hsrω 66 Hsp90GFP larvae, but not in hsrω 66 or Hsp90GFP single mutants. Hsp83 binds at these gene promoters. Several omega speckle associated hnRNPs too may bind with these genes and transcripts. Hsp83-hnRNP interactions are also known. Thus, elevated Hsp83 in altered hnRNP distribution and dynamics, following absence of hsrω lncRNAs and omega speckles, background can severely perturb regulatory circuits with unexpected consequences, including down-regulation of tumor suppressor gene like l(2)gl. 0 1 5