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.