Altered levels of hsromega lncRNAs further enhance Ras signaling during ectopically activated Ras induced R7 differentiation in Drosophila

Abstract

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..

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

“…17,18 Therefore, to know reasons for the reduced levels of ecdysone noted above in early dying pupae, we next examined Dilp8 protein levels in eye discs with normal (sev-GAL4>UAS-GFP), high (sev-GAL4>UAS-Ras1 V12 ), and very high (sev-GAL4>UAS-Ras1 V12 hsrω-RNAi) Ras activity. 11 Immunostaining of late third instar larval eye discs with Dilp8 antibody 17 revealed absence of Dilp8 staining in the ommatidial cells ( Figure 4A,B,E,F,I,J) but a faint presence of Dilp8 in cytoplasm of peripodial cells in all the three genotypes ( Figure 4C,D,G,H,K,L). The Dilp8 staining in ommatidial cells of 8 to 9 hours pupal eye discs coexpressing sev-GAL4>UAS-Ras1 V12 and hsrω-RNAi was slightly enhanced ( Figure 4U,V), but their peripodial cells showed much stronger levels ( Figure 3W,X) than their same age counterparts in sev-GAL4>UAS-GFP ( Figure 4M-P) or sev-GAL4>UAS-Ras1 V12 (Figure 4Q-T).…”

“…Some of these seem to be primarily due to activated Ras or altered hsrω expression since the Ras/MAPK pathway is known to regulate immune response 32,33 while the hsrω transcripts have roles in stress responses, cell death, and several other pathways. 5,8,11,34,35 However, beside the predicted upregulation of G-protein coupled signal transduction pathway following ectopic expression of activated Ras, a significant increase in expression of several JNK pathway genes was also noted (Tables 2 and 3).…”

“…28 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, 29 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. 11 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. 11 Upregulation of Ras activity further in sev-GAL4>UAS-Ras1 V12 expressing eye discs 11 by co-expressing hsrω-RNAi transgene 13 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 .…”

“…29 Scale bar in F denotes 50 μm and applies to E-P. Q and R, Confocal and DIC images of parts of DAPI-stained (white) salivary glands (SG, Q) and Malpighian tubules (MT, R) in 24-to 25-hour-old GMR-GAL4>UAS-Ras1 V12 pupae. S, Schematics of cells with varying levels of activated Ras in ommatidial units in eye discs of the indicated genotypes 11 24-to 25-hour-old sev-GAL4>UAS-Ras1 V12 hsrω-RNAi pupae. They continued to show these structures as in 8-to 9-hour-old pupae even at 24 to 25 hours APF ( Figure 1G,J, M,P).…”

“…The GMR-GAL4>UAS-GFP expressing dorsomedian paired neurons in ventral ganglia (VG) also persisted in 24-to 25-hour-old GMR-GAL4>UAS-Ras1 V12 pupae (not shown). A cartoon showing the varying levels of activated Ras in eye discs of different genotypes, based on our earlier results, 11 is presented in Figure 1S. It is significant that absence of metamorphic changes during development of pupa and the early pupal death (~25-30 hours APF) in GMR-GAL4>UAS-Ras1 V12 and sev-GAL4>UAS-Ras1 V12 hsrω-RNAi genotypes correlates with the earlier reported 11 wider and stronger Ras signaling in their eye discs than in the sev-GAL4>UAS-Ras1 V12 eye discs.…”