Genome-wide RNAi analysis of JAK/STAT signaling components in Drosophila

Abstract

The cytokine-activated Janus kinase (JAK)/signal transducer and activator of transcription (STAT) pathway plays an important role in the control of a wide variety of biological processes. When misregulated, JAK/STAT signaling is associated with various human diseases, such as immune disorders and tumorigenesis. To gain insights into the mechanisms by which JAK/STAT signaling participates in these diverse biological responses, we carried out a genome-wide RNA interference (RNAi) screen in cultured Drosophila cells. We identified 121 genes whose double-stranded RNA (dsRNA)-mediated knockdowns affected STAT92E activity. Of the 29 positive regulators, 13 are required for the tyrosine phosphorylation of STAT92E. Furthermore, we found that the Drosophila homologs of RanBP3 and RanBP10 are negative regulators of JAK/STAT signaling through their control of nucleocytoplasmic transport of STAT92E. In addition, we identified a key negative regulator of Drosophila JAK/STAT signaling, protein tyrosine phosphatase PTP61F, and showed that it is a transcriptional target of JAK/STAT signaling, thus revealing a novel negative feedback loop. Our study has uncovered many uncharacterized genes required for different steps of the JAK/STAT signaling pathway.

Figure 1.

Generating a JAK/STAT reporter construct. (A) Schematic representation of the 10XSTAT92E–luciferase reporter construct. Five copies of a genomic fragment from the SOCS36E intronic region containing two STAT92E-binding sites were placed upstream of a hsp minimal promoter-driven firefly luciferase gene. (B) Drosophila S2-NP cells were transfected with 10XSTAT92E–luciferase and Act-Renilla together with dsRNAs against various canonical components of the JAK/STAT pathway. Luciferase assay was performed 4 d later, and the reporter activity was normalized as the ratio of firefly/Renilla. Note that the control value was set to 1. The results were from two independent experiments.

Figure 2.

Data analysis for the JAK/STAT screen. (A) Scatter plot for three representative screen plates. Cutoffs were set as 2 SD below the mean or 3 SD above the mean RLU. Note that all three “spiked in” control dsRNAs against STAT92E were identified. (B) Pie chart depicting categories of genes identified in the JAK/STAT screen.

Figure 3.

Identification of genes required for Upd-induced tyrosine phosphorylation of STAT92E. (A) Act-STAT92E-HA was transfected into S2-NP cells together with dsRNA against lacZ. Cells were split into two dishes 3.5 d after transfection. Half of the cells were cocultured with S2-NP cells transfected with Act-Upd ∼12 h prior to harvest and the other half remained untreated. Cell extracts were subjected to immunoprecipitation using anti-HA antibodies and the immunoprecipitates were analyzed by immunoblotting using anti-phospho-Tyr-STAT92E and HA antibodies. Note that Upd-induction leads to a dramatic increase in STAT92E phosphorylation. (B) Act-STAT92E-HA was transfected into S2-NP cells together with various dsRNAs targeting positive regulators identified in the screen. Cells were stimulated with Upd ∼12 h prior to harvest. Cell extracts were subjected to Western blot analysis using anti-phospho-Tyr-STAT92E and HA antibodies. dsRNAs against LacZ and lilli serve as control. (C) List of genes required for Upd-induced STAT92E phosphorylation.

Figure 4.

Drosophila homologs of RanBP3 (CG11763) and RanBP10 (CG10225) are involved in phosphorylated STAT92E nucleocytoplasmic shuttling. Cells were treated with various dsRNAs and then transfected with Act-Upd 4 d later (G–O) or remained untransfected (A–F). Immunostaining was performed using anti-phospho-Tyr-STAT92E antibody (green). DAPI staining was employed to visualize the nuclei (red). Note that a significant accumulation of phosphorylated STAT92E in the nuclei of cells treated with dsRNA against either CG11763 or CG10225 was detected upon Upd induction, compared with cells treated with dsRNA for lacZ.

Figure 5.

PTP61F negatively regulates the JAK/STAT pathway in Drosophila. (A) Knockdown of PTP61F by RNAi activates the JAK/STAT reporter activity. Drosophila S2-NP cells were transfected with 10XSTAT92E–luciferase and Act-Renilla together with dsRNAs against lacZ or PTP61F. Luciferase assay was performed 4 d later and the reporter activity was normalized as the ratio of firefly to Renilla. The control value was set as 1. The results were from two independent experiments. (B) Act-Myc-Hop was transfected into S2-NP cells together with dsRNAs against lacZ or PTP61F. Cells were harvested and cell lysates were immunoprecipitated with anti-Myc antibody. Immunoprecipitates were analyzed by immunoblotting using anti-phospho-Tyr or anti-Myc antibodies. Note that an increase in phospho-Hop levels was detected upon RNAi knockdown of PTP61F. (C) Act-STAT92E-HA was transfected into S2-NP cells together with dsRNAs against lacZ or PTP61F. Cells were harvested and cell lysates were immunoprecipitated with anti-HA antibody. Immunoprecipitates were analyzed by immunoblotting using anti-phospho-Tyr-STAT92E or anti-HA antibodies. An increase in phospho-STAT92E levels was detected upon RNAi knockdown of PTP61F. (D) RNA in situ hybridization using STAT92E or PTP61F probes was performed on wild-type stage 9–10 embryos (panels a,b), embryos overexpressing Upd under the control of paired-Gal4 (panel c), and hop GLC embryos (panel d). Note that PTP61F transcript levels are dramatically increased in the paired domain (panel c) and decreased in embryo lacking hop activity (panel d). (E) Genetic interactions between Upd and PTP61F. Overexpression of Upd in the eye under the control of GMR-Gal4 results in a dramatic overgrowth and deformation in the adult eye (cf. panels a and b). Removing one copy of PTP61F further enhances this phenotype (panel c), whereas introduction of a PTP61F transgene rescues this phenotype (panel d).