Abstract
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 in vivo, 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 prpf19 and histone modifier rnf2; based on their known roles in developmental processes, we further investigated these two genes. We found that prpf19 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 cbx and phc RNAi planarians. To identify genes regulated by PRC1, we performed RNA-seq after knocking down rnf2 or phc. Although these proteins are predicted to function in the same complex, we found that the set of genes differentially expressed in rnf2 versus phc RNAi were largely non-overlapping. Using in situ hybridization, we showed that rnf2 regulates gene expression levels within a tissue type, whereas phc is necessary for the spatial restriction of gene expression, findings consistent with their respective in vivo 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.
Highlights
A deep understanding of the networks and signaling pathways that direct the maintenance and differentiation of adult stem cells is essential for regenerative therapies
We found several to be essential for homeostatic maintenance, regeneration, and tissue patterning, including spliceosomal factor prpf19 and epigenetic factors rnf2 and bre1, known to ubiquitylate histones H2A and H2B, respectively. prpf19 was required for worm survival but not for stem cell maintenance, suggesting a role in promoting cell differentiation
To examine if the effects of prpf19 RNAi were being mediated through disruption of a conserved spliceosomal complex, we knocked down three homologs of core NineTeen Complex (NTC) component members, cdc5l, plrg1, and spf27 (Supplementary Table S3). We found that these genes were necessary for worm survival and regeneration (Figures 4B,C). cdc5l and plrg1 are essential for NTC function in yeast and presented very severe phenotypes in S. mediterranea, with RNAi animals phenocopying the head regression, ventral curling, and lysis that we observed after prpf19 RNAi. spf27(RNAi) worms displayed a milder phenotype than other NTC genes examined and showed delayed or absent regeneration in 28/37 head fragments and 33/37 trunk fragments; an additional three trunk fragments showed a more severe ventral curling and lysis phenotype
Summary
A deep understanding of the networks and signaling pathways that direct the maintenance and differentiation of adult stem cells is essential for regenerative therapies. The freshwater planarian, Schmidtea mediterranea, is an important model for studying the molecular mechanisms that underpin stem cell-based regeneration (Elliott and Sánchez Alvarado, 2013; Ivankovic et al, 2019). These worms maintain a large population of adult stem cells, a subset of which have been demonstrated to be pluripotent (Baguñà et al, 1989; Wagner et al, 2011). This population of stem cells continuously renews planarian tissues during homeostasis and is mobilized in response to injury to regenerate tissues (Saló and Baguñà, 1985; Abnave et al, 2017). One essential post-translation regulator of proteins is the addition of the small, highly conserved polypeptide ubiquitin, which modifies protein function in myriad cellular contexts, including transcription, cell cycle regulation, translational fidelity, protein turnover, and degradation (Ciechanover et al, 1984; Nakayama and Nakayama, 2005; Endoh et al, 2012; Higgins et al, 2015)
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