Modulation of the Hippo pathway and organ growth by RNA processing proteins

Abstract

The Hippo tumor-suppressor pathway regulates organ growth, cell proliferation, and stem cell biology. Defects in Hippo signaling and hyperactivation of its downstream effectors-Yorkie (Yki) in Drosophila and YAP/TAZ in mammals-result in progenitor cell expansion and overgrowth of multiple organs and contribute to cancer development. Deciphering the mechanisms that regulate the activity of the Hippo pathway is key to understanding its function and for therapeutic targeting. However, although the Hippo kinase cascade and several other upstream inputs have been identified, the mechanisms that regulate Yki/YAP/TAZ activity are still incompletely understood. To identify new regulators of Yki activity, we screened in Drosophila for suppressors of tissue overgrowth and Yki activation caused by overexpression of atypical protein kinase C (aPKC), a member of the apical cell polarity complex. In this screen, we identified mutations in the heterogeneous nuclear ribonucleoprotein Hrb27C that strongly suppressed the tissue defects induced by ectopic expression of aPKC. Hrb27C was required for aPKC-induced tissue growth and Yki target gene expression but did not affect general gene expression. Genetic and biochemical experiments showed that Hrb27C affects Yki phosphorylation. Other RNA-binding proteins known to interact with Hrb27C for mRNA transport in oocytes were also required for normal Yki activity, although they suppressed Yki output. Based on the known functions of Hrb27C, we conclude that Hrb27C-mediated control of mRNA splicing, localization, or translation is essential for coordinated activity of the Hippo pathway.

Keywords: Hippo pathway; Hrb27C; RNA-binding proteins; aPKC; hnRNP.

Fig. 1.

Hrb27C mutations are dominant suppressors of the aPKC overexpression phenotype in the Drosophila eye. (A and B) Confocal images of wing imaginal discs from third instar larvae expressing GFP (green or gray) in a stripe of cells along the anterior-posterior compartment boundary driven by dpp-Gal4 and white-RNAi (A) or aPKCζ* and white-RNAi (B). (C) Eye of a fly with one copy of the GMR-Gal4 transgene. (D) Eye of a fly with GMR-Gal4–driven overexpression of aPKCζ*. (E–G) GMR>aPKCζ* flies also carrying one copy of the allele Hrb27C^J6, Hrb27C^F2-1, or Df(2L)BSC108 (which deletes Hrb27C). (H) GMR>aPKCζ* fly coexpressing Hrb27C-RNAi (v16041). (I) Diagram of the Hrb27C protein depicting its functional domains and the location of the Hrb27C^F2-1 and Hrb27C^J6 mutations. The gray bars at the C-terminal end mark the locations of epitopes recognized by the Hrb27C polyclonal antibodies. RRM, RNA-recognition motif.

Fig. 2.

Loss of Hrb27C fucntion suppresses Yki activity. (A–Q) Confocal images of wing imaginal discs from third instar larvae expressing GFP (green or gray) in a stripe of cells along the anterior-posterior compartment boundary driven by dpp-Gal4 (A and B) or ptc-Gal4 (J–O), or in the posterior compartment driven by hh-Gal4 (C–I, P, and Q). In addition, discs expressed Hrb27C-RNAi (A, B, D, G, I, K, M, O, Q), Hrb27C-FLAG (E), and aPKCζ* and Hrb27C-RNAi (B). Discs were stained for β-galactosidase to detect ex-lacZ (ExZ), diap1-lacZ (diap1Z), myc-lacZ (MycZ), or Dll-lacZ (DllZ) expression, and Cut, Wg, or Cubitus interruptus (Ci), as indicated (red or gray). (R) Firefly luciferase expression levels of S2 cells transiently expressing Yki-Gal4DBD or Sc-Gal4DBD together with UAS-luc. Firefly expression levels were normalized against a constitutive Renilla luciferase. Cells were transfected with dsRNAs targeting GFP, Hrb27C, wts, or Spase25.

Fig. 3.

Hrb27C is required for tissue growth. (A) Hrb27C^F2-1/Df(2L)Exel17029 (Right) and a Df(2L)Exel17029/CyO-RFP sibling (Left) third instar larva of the same age. Bright field picture (A) and RFP expression from the CyO-RFP balancer chromosome (A′). (B and C) Third instar eye discs with wild-type (wt) clones (B) and homozygous Hrb27C^J6 mutant clones (C) (black, indicated with arrowheads) flipped against ubi-GFP. (D and E) Eyes of flies in which a wild-type chromosome (D) or a Hrb27C^J6 mutant chromosome (E) was flipped against a w+ marked chromosome with a cell lethal mutation. Homozygous wild-type or Hrb27C^J6 homozygous cells are light orange, and heterozygous cells are red. While wild-type cells make big patches, Hrb27C^J6 homozygous cells form only small patches and most of the eye is composed of heterozygous red cells. (F) Eye of a fly with an ey-Gal4 driver (control). (G) Eye of a fly with ey-Gal4–driven Hrb27C-RNAi expression. (H and I) Eye discs with positively GFP-marked clones expressing Hrb27C-RNAi generated by MARCM stained for BrdU incorporation (red or gray, indicated with arrowheads) (H) or Cyclin E (CycE; red or gray, indicated with arrowheads) (I) . (J) Third instar eye disc with Hrb27C^J6 mutant clones marked by the lack of GFP expression and stained for Sens (red or gray) and the neuronal marker ELAV (blue or gray).

Fig. 4.

Hrb27C intersects the Hippo pathway between Wts and Yki. (A–N) Wing imaginal discs expressing ptc-Gal4, UAS-GFP (green) plus transgenes as indicated on the left, together with white-RNAi (A, C, E, G, I, K, and M) or Hrb27C-RNAi (B, D, F, H, J, L, and N). Discs were stained to detect the expression of the ex-lacZ reporter (ExZ; red or gray) and nuclei (DAPI; blue). Presumptive pouch regions are shown except for G and H, which show the notum regions, because Sd^GA impaired pouch development, likely by disrupting normal Sd function that is required for wing development independent of Yki. (O) Wing imaginal discs expressing ptc-Gal4, UAS-GFP, Hrb27C-RNAi stained to detect Yki (red or gray) and nuclei (DAPI; blue). (P) Western blot showing the effect of Hrb27C knockdown on Yki phosphorylation in combination with other genetic manipulations, as indicated.

Fig. 5.

Yki regulation by RNA-binding proteins. Wing imaginal discs expressing hh-Gal4, UAS-GFP (green) plus sqd-RNAi (A), glo-RNAi (C), Pabp2-RNAi (E), hfp-RNAi (G), and in combination with Hrb27C-RNAi (B, D, F, and H, respectively). Discs were stained to detect the expression of the ex-lacZ reporter (ExZ; red or gray) and nuclei (DAPI; blue).