A genome-wide screen for peroxisome regulators connects peroxisomes with Wnt signaling through RNF146 and tankyrase [Amplicon-sequencing]

Peroxisomes are membrane-bound organelles that help cells specialize their metabolism. In humans, peroxisomes are required for normal development, yet the genes regulating peroxisome function remain unclear. Thus, we performed a genome-wide CRISPRi screen to identify novel factors involved in peroxisomal homeostasis. We found that transcriptional inhibition of RNF146, an E3 ligase activated by poly(ADP-ribose), dramatically reduced the import of proteins into peroxisomes. The observed RNF146-mediated loss of peroxisome import depended on the stabilization and activity of the poly(ADP-ribose) polymerase tankyrase, which binds the peroxisomal membrane protein PEX14. We propose a model in which RNF146 and tankyrase regulate peroxisome import efficiency by tuning PARsylation of proteins at the peroxisome membrane. Interestingly, we found that perturbations to peroxisomes altered tankyrase's selection of substrates, including the beta-catenin destruction complex component AXIN1. The loss of peroxisomes caused tankyrase and RNF146-dependent degradation of AXIN1 and a concomitant increase of beta-catenin transcription. Together, these observations not only suggest previously undescribed roles for RNF146 in peroxisomal regulation, but also a novel role in bridging peroxisome function with Wnt/beta-catenin signaling during development. Overall design: We performed a genome-wide CRISPRi screen for genes that regulate peroxisome formation and maintenance. The starting point is a Pex-ZeoR cell line, in which the efficiency of peroxisome import is linked to cell viability by fusing the fluorescent marker mVenus and a peroxisomal targeting signal (PTS1) to the gene encoding resistance to Zeocin. Cells with functional peroxisomes sequester the Zeocin resistance protein (ZeoR), thereby preventing them from neutralizing Zeocin. By contrast, cells with reduced peroxisome import should accumulate mVenus-ZeoR-PTS1 in the cytoplasm where it can neutralize Zeocin, conferring a selective advantage in the presence of Zeocin. Knockdown of genes that promote peroxisomes should increase viability when challenged with Zeocin. Knockdown of genes that inhibit peroxisome formation should decrease viability when challenged with Zeocin.