Altered levels ofhsromegalncRNAs further enhance Ras signaling during ectopically activated Ras induced R7 differentiation inDrosophila

Abstract

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.

Activated Ras/JNK driven Dilp8 in imaginal discs adversely affects organismal homeostasis during early pupal stage in Drosophila, a new checkpoint for development

“…We had earlier found a genetic interaction between Hsp83, Ras signaling pathways and the hsrω lncRNAs . Confirming these earlier studies, more recently we found that the ectopically induced high level of Ras signaling cascade was further enhanced when hsrω lncRNA levels were simultaneously downregulated in eye discs of Drosophila third instar larvae . This study showed that while the sev‐GAL4 driven ectopic expression of activated Ras in developing eye discs led to substantial late pupal death as pharates, still higher levels of activated Ras in eye discs, achieved by co‐expression of sev‐GAL4 driven activated Ras and hsrω‐RNAi caused early pupal death (~25‐30 hours after pupation).…”

“…The observed pupal death following ectopic expression of certain transgenes driven by predominantly eye‐specific sev‐GAL4 or GMR‐GAL4 drivers in our and several other earlier studies has been intriguing since eyes are not essential for survival of flies . In this study, therefore, we have examined possible reasons for the early pupal death following induction of very high levels of activated Ras in larval imaginal discs.…”

“…UAS‐Ras1 V12 transgene expression has been widely used for sustained Ras signaling since the Ras1 V12 protein does not require upstream events for activation . In this study, we expressed the activated Ras1 V12 protein in eye disc cells using GMR‐GAL4 or sev‐GAL4 drivers, the former driving expression in a much greater number of cells in eye discs than the later, and accordingly the Ras signaling is much more widespread in eye discs expressing the GMR‐GAL4 (not shown) than in those expressing the sev‐GAL4 driver . Correspondingly, all the GMR‐GAL4 > UAS‐Ras1 V12 individuals died at early pupal stage (between 25 and 30 hours APF), whereas the sev‐GAL4 > UAS‐Ras1 V12 pupae developed normally till late pupal stages but a majority (~88%, N = 1058) died as pharates while the rest eclosed with rough eyes due to extra R7 photoreceptors .…”

“…In this study, we expressed the activated Ras1 V12 protein in eye disc cells using GMR‐GAL4 or sev‐GAL4 drivers, the former driving expression in a much greater number of cells in eye discs than the later, and accordingly the Ras signaling is much more widespread in eye discs expressing the GMR‐GAL4 (not shown) than in those expressing the sev‐GAL4 driver . Correspondingly, all the GMR‐GAL4 > UAS‐Ras1 V12 individuals died at early pupal stage (between 25 and 30 hours APF), whereas the sev‐GAL4 > UAS‐Ras1 V12 pupae developed normally till late pupal stages but a majority (~88%, N = 1058) died as pharates while the rest eclosed with rough eyes due to extra R7 photoreceptors . Upregulation of Ras activity further in sev‐GAL4>UAS‐Ras1 V12 expressing eye discs by co‐expressing hsrω‐RNAi transgene that downregulates the hsrω lncRNAs, resulted in early pupal death of all sev‐GAL4 > UAS‐Ras1 V12 hsrω‐RNAi like that in the GMR‐GAL4 > UAS‐Ras V12 .…”

“…Correspondingly, all the GMR‐GAL4 > UAS‐Ras1 V12 individuals died at early pupal stage (between 25 and 30 hours APF), whereas the sev‐GAL4 > UAS‐Ras1 V12 pupae developed normally till late pupal stages but a majority (~88%, N = 1058) died as pharates while the rest eclosed with rough eyes due to extra R7 photoreceptors . Upregulation of Ras activity further in sev‐GAL4>UAS‐Ras1 V12 expressing eye discs by co‐expressing hsrω‐RNAi transgene that downregulates the hsrω lncRNAs, resulted in early pupal death of all sev‐GAL4 > UAS‐Ras1 V12 hsrω‐RNAi like that in the GMR‐GAL4 > UAS‐Ras V12 . Normally, the pupae undergo head eversion after 12 hours of pupation so that the 12 to 14 hours APF stage wild‐type pupae show a proper demarcation of head, thorax, and abdomen (Figure A).…”