UC Santa Barbara

Peroxisomes are compartmentalized membrane-bound organelles evolved to enable cells to metabolically adapt to their environment, that are themselves near omnipresent throughout eukarya. In humans, peroxisomes are essential for normal development, insofar that peroxisome dysfunction causes both pre- and post-natal mortal insufficiencies that would disbar one from addressing the vicissitudes of life; and yet there is a dearth of data detailing the genes governing peroxisome function and homeostasis. To this end, we executed a genome-wide CRISPRi screen to identify novel factors involved in peroxisomal homeostasis. We uncovered various genes that hold potential as novel regulators of peroxisomes, such as suppressor of cytokine signaling-3 (SOCS3), C2orf15, Insulin Degrading Enzyme (IDE), and Ring finger protein 146 (RNF146). Through rigorous inspection, we were able to classify SOCS3 as a spurious hit generated by off-target effects, while potentially developing a novel way to find Cas9 related off-targets. Contemporaneously, we found through study of C2orf15 that there were computational aberrations embedded in the sgRNA generation processes of our colleagues’ CRISPRi library, leading us to the discovery that Mitochondrial Ribosomal Protein L30 (MRPL30) modulates peroxisome abundance. Our dual-screen experiments then showed that IDE resembled a profligate peroxisome cargo regulator where its suppression strongly bolstered peroxisome matrix import, an antipodal effect relative to most peroxisome biogenesis gene knockdowns. We chose to focus our efforts on RNF146, an E3 ligase with a predilection for poly(ADP-ribose), where its suppression caused an ebbing in the import function of peroxisomes. Loss of RNF146 caused peroxisome import malfunction by unfettered oversaturation of the poly(ADP-ribose) polymerases TNKS and TNKS2, which then bind, PARsylate, and impair peroxisomal proteins, chiefly PEX14. Because RNF146/TNKS/2 are more renowned for their connection to Wnt-signaling, mainly the degradation of AXIN1, a component of the destruction complex, we hypothesized that the peroxisome, via PEX14, would participate in the homeostatic equilibrium of RNF146/TNKS/2’s proteasomal facilitations. We discovered that both loss of PEX14 and PEX19 destabilized AXIN1, as predicted, which subsequently compromised the steady-state levels of beta-catenin induced transcription. We then extrapolated these findings to other realms of developmental biology, mainly the Wnt-signaling influenced stem cell fate of neural progenitor cells (NPCs) and neural crest stem cells (NCSCs). Interestingly, our early results suggest that loss of PEX14 and PEX19 may bias the differentiation decision of stem cells similar to a loss in AXIN1, in line with our model. In totality, the consequences of our genome-wide screen, the collective observations, and various data paint a larger picture in which peroxisomes are interlinked to, and participate in, multifarious regulatory pathways of the cell.