Pkc53E mediates miR-316-dependent suppression of Yorkie-driven overgrowth

The Hippo pathway restricts tissue growth by inhibiting the transcriptional co-activator Yorkie (Yki), and aberrant Yki activation drives tissue overgrowth in Drosophila. While kinase-dependent regulation of Yki has been intensively studied, how microRNAs modulate Yki-driven growth programs in vivo remains less clear. Here, we conducted an in vivo modifier screen of 145 Drosophila microRNAs using two Yki-driven eye overgrowth models (GMR > Yki^WT and GMR > Yki^S168A). We identified multiple miRNAs that significantly enhanced or suppressed Yki-induced overgrowth, and focused on miR-316 as a robust suppressor whose inhibition by a miRNA sponge showed the opposite effect. Integrative target prediction followed by RNAi-based functional screening nominated Pkc53E, a conventional protein kinase C and the Drosophila ortholog of mammalian PKCα, as a functional downstream effector of miR-316. Pkc53E knockdown phenocopied miR-316-mediated suppression, reducing Yki-driven eye overgrowth and mitotic activity, whereas Pkc53E overexpression enhanced growth. In an imaging-optimized wing system, depletion of Pkc53E reduced wing growth and lowered the protein levels of canonical Yki targets, including CycE, DIAP1, and Expanded. Notably, miR-316 overexpression was associated with increased Yki phosphorylation. Together, our results identify a miR-316-Pkc53E axis that modulates Yki-dependent proliferation and growth, revealing a microRNA-mediated layer of post-transcriptional control that fine-tunes Hippo pathway output in vivo.