Fig. 4: Mahe directly binds to hop transcripts and increases/stabilizes its level leading to upregulation of upd2 ligand. | Cell Death & Disease

Fig. 4: Mahe directly binds to hop transcripts and increases/stabilizes its level leading to upregulation of upd2 ligand.

From: Maheshvara regulates JAK/STAT signaling by interacting and stabilizing hopscotch transcripts which leads to apoptosis in Drosophila melanogaster

Fig. 4

a, b, d RNA immunoprecipitation was done using Anti-HA beads to immunoprecipitate HA-tagged-Mahe along with associated RNA-protein complex. a Protein lysates from GMR-GAL4 > UAS-HA-mahe was used for immunoprecipitation followed by RT-PCR with primers specific for components of JAK/STAT pathway. Out of the different components upd1, upd2, upd3, dome, hop,stat92E, and socs36E of JAK/STAT signaling, only hop was amplified indicating that Mahe binds with hop transcripts. b No amplification was observed in negative control in which GMR-GAL4/+ lysate was used for immunoprecipitation. c cDNA samples without RNA immunoprecipitation were used for positive control. d No amplification was observed with primer specific for hop promoter, while hop exon specific primers showed positive amplification. eg RNA-FISH was done to check the level of hop transcripts in eye antennal discs. e FISH with hop-specific antisense riboprobe in wild-type eye antennal disc showed a very little signal. f Ectopic mahe expression revealed enhanced levels of hop transcript posterior to the morphogenetic furrow when compared to that of the control (area in rectangle). g hop sense riboprobe was used as negative control and no signal was seen in mahe overexpressed eye-antennal discs. h Graph represents hop intensity per unit area that clearly shows mahe overexpression leads to increase in hop transcript levels when compared to that of wild-type control. One-way ANOVA. i Quantitative real-time PCR shows increase in hop transcripts level upon mahe overexpression when compared to transcripts from GMR-GAL4 control tissue. One-way ANOVA. j Enrichment of both hop pre-mRNA and mRNA was observed by real-time PCR in RIP precipitate. One-way ANOVA. k Overexpression of hop alone leads to increase in upd2 transcript levels, similar to that of ectopic mahe. One-way ANOVA. Scale bar in 50 μm (eg). Genotypes (b, c, e, h, i, j, k) w/+; +; GMR-GAL4/+ (a, d, f, g, h, i, j, k) w/+; +; GMR-GAL4,UAS-HA-mahe/UAS-HA-mahe (i, k) w/+; UAS-mahe-RNAi/+; GMR-GAL4/+ (k) w/+; UAS-hop/+; GMR-GAL4/+ (k) w/+; UAS-hop-RNAi/+; GMR-GAL4/+. One-way ANOVA * P < 0.05, ** P < 0.01, *** P < 0.001.

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