RNAi Screen of RING/U-Box Domain Ubiquitin Ligases Identifies Critical Regulators of Tissue Regeneration in Planarians.

Regenerative processes depend on the interpretation of signals to coordinate cell behaviors. The role of ubiquitin-mediated signaling is known to be important in many cellular and biological contexts, but its role in regeneration is not well understood. To investigate how ubiquitylation impacts tissue regeneration , we are studying planarians that are capable of regenerating after nearly any injury using a population of stem cells. Here we used RNAi to screen RING/U-box E3 ubiquitin ligases that are highly expressed in planarian stem cells and stem cell progeny. RNAi screening identified nine genes with functions in regeneration, including the spliceosomal factor and histone modifier ; based on their known roles in developmental processes, we further investigated these two genes. We found that was required for animal survival but not for stem cell maintenance, suggesting a role in promoting cell differentiation. Because RNF2 is the catalytic subunit of the Polycomb Repressive Complex 1 (PRC1), we also examined other putative members of this complex (CBX and PHC). We observed a striking phenotype of regional tissue misspecification in and RNAi planarians. To identify genes regulated by PRC1, we performed RNA-seq after knocking down or Although these proteins are predicted to function in the same complex, we found that the set of genes differentially expressed in versus RNAi were largely non-overlapping. Using hybridization, we showed that regulates gene expression levels within a tissue type, whereas is necessary for the spatial restriction of gene expression, findings consistent with their respective phenotypes. This work not only uncovered roles for RING/U-box E3 ligases in stem cell regulation and regeneration, but also identified differential gene targets for two putative PRC1 factors required for maintaining cell-type-specific gene expression in planarians.