Journal: Genes & Development
Article Title: Hyd/UBR5 defines a tumor suppressor pathway that links Polycomb repressive complex to regulated protein degradation in tissue growth control and tumorigenesis
doi: 10.1101/gad.351856.124
Figure Lengend Snippet: Identification of Hyd as a critical E3 ubiquitin ligase mediating Lin-induced Bowl degradation. ( A ) Schematic diagram of a pooled RNAi screen for E3 ligases required for Lin-induced Bowl degradation. S2R + cells transfected with HA-Bowl and FLAG-Lin were split into 24 well plates, each well treated with a pool of dsRNAs targeting four different E3 ligases, followed by Western blot analysis of cell lysates. ( B ) Confirmation of Hyd as an E3 ligase required for Lin-induced Bowl degradation. S2R + cells expressing HA-Bowl and FLAG-Lin were treated with different Hyd dsRNAs. (Hyd-1) dsRNA in the original RNAi library (Hyd-2, Hyd-3, and Hyd-4) three synthesized dsRNAs targeting different regions of Hyd. HA-Bowl was normally undetectable under such conditions due to Lin-induced degradation. However, it was stabilized by RNAi against Hyd or Lin. GFP dsRNA was included as a negative control. ( C ) S2R + cells expressing the indicated constructs were subjected to coimmunoprecipitation (co-IP) assay as indicated. Cells were treated with 10 μM PS-341 for 4 h before harvesting. Interaction was readily detected between FLAG-Lin and HA-Bowl (lane 3 ) and between FLAG-Lin and Hyd-HA (lane 4 ). (Lane 5 ) Neither pairwise interaction was affected by coexpression of the third protein. ( D ) S2R + cells expressing the indicated constructs were subjected to co-IP assay as indicated. Cells were treated with PS-341 as in C . Co-IP between FLAG-Bowl and Hyd-HA was detected only in the presence of Myc-Lin (cf. lanes 2 and 5 ). ( E ) S2R + cells expressing the indicated constructs were subjected to co-IP assay as indicated. (HA-Bowl 122–373K137R ) The K137R mutant form of Bowl fragment 122–373 as described in C. Note that the Lin–Bowl co-IP was severely impaired by lin A25 , but not lin A24 , mutation. ( F ) S2R + cells expressing the indicated constructs were subjected to co-IP assay as indicated. Note that the Lin–Hyd co-IP was impaired by both lin A24 and lin A25 mutations. ( G ) S2R + cells expressing the indicated constructs were subjected to co-IP assay as indicated. Cells were treated with PS-341 as in C . Note the disruption of the Lin–Bowl interaction by Drm (cf. lanes 2 and 3 ). ( H ) S2R + cells expressing the indicated constructs were treated with PS-341 before IP with FLAG antibody. The IP product was probed with antiubiquitin antibody to detect Bowl ubiquitination. Lin-induced Bowl ubiquitination was suppressed by Drm. ( I ) Schematic model for the regulation of Bowl degradation. In the absence of Drm, Lin promotes Bowl degradation by functioning as a substrate adaptor protein recruiting Bowl to Hyd. Drm competes with Bowl for Lin binding, thus releasing Bowl from Lin–Hyd, resulting in Bowl stabilization.
Article Snippet: For the dsRNA screen, the DNA templates of dsRNAs from Drosophila RNAi library (Open Biosystem) were PCR-amplified and transcribed with 5XMEGAScript T7 kit (Ambion).
Techniques: Transfection, Western Blot, Expressing, Synthesized, Negative Control, Construct, Co-Immunoprecipitation Assay, Mutagenesis, Disruption, Binding Assay
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