Genome-wide suppressor screen identifies USP35/USP38 as therapeutic candidates for ciliopathies
I-Chun Tsai, … , Perciliz L. Tan, Nicholas Katsanis
I-Chun Tsai, … , Perciliz L. Tan, Nicholas Katsanis
Published November 14, 2019
Citation Information: JCI Insight. 2019;4(22):e130516. https://doi.org/10.1172/jci.insight.130516.
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Research Article Genetics Therapeutics

Genome-wide suppressor screen identifies USP35/USP38 as therapeutic candidates for ciliopathies

Abstract

The ciliopathies are a group of phenotypically overlapping disorders caused by structural or functional defects in the primary cilium. Although disruption of numerous signaling pathways and cellular trafficking events have been implicated in ciliary pathology, treatment options for affected individuals remain limited. Here, we performed a genome-wide RNAi (RNA interference) screen to identify genetic suppressors of BBS4, one of the genes mutated in Bardet-Biedl syndrome (BBS). We discovered 10 genes that, when silenced, ameliorate BBS4-dependent pathology. One of these encodes USP35, a negative regulator of the ubiquitin proteasome system, suggesting that inhibition of a deubiquitinase, and subsequent facilitation of the clearance of signaling components, might ameliorate BBS-relevant phenotypes. Testing of this hypothesis in transient and stable zebrafish genetic models showed this posit to be true; suppression or ablation of usp35 ameliorated hallmark ciliopathy defects including impaired convergent extension (CE), renal tubule convolution, and retinal degeneration with concomitant clearance of effectors such as β-catenin and rhodopsin. Together, our findings reinforce a direct link between proteasome-dependent degradation and ciliopathies and suggest that augmentation of this system might offer a rational path to novel therapeutic modalities.

Authors

I-Chun Tsai, Kevin A. Adams, Joyce A. Tzeng, Omar Shennib, Perciliz L. Tan, Nicholas Katsanis

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Figure 3

The molecular mechanism by which USP35 suppression ameliorate the defects caused by BBS4 depletion.

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The molecular mechanism by which USP35 suppression ameliorate the defect...
(A) Suppression of USP35 enhances the ubiquitination of β-catenin. BBS4-depleted HEK293 cells were transfected with either shRNA-control or shRNA targeting to USP35. 72 hours after transfection, immunoprecipitation of β-catenin was performed, followed by immunoblotting with β-catenin and ubiquitin. The relative quantification of β-catenin ubiquitination was performed with QuantityOne. *P < 0.05, **P < 0.01 (2-tailed Student’s t test). (B) USP35 suppression facilitated the degradation of accumulated NICD. HEK293 cells were transfected with the plasmids as indicated in the figure. Seventy-two hours after transfection, cells were harvested and subjected to immunoblotting of NICD and GAPDH (loading control). Relative levels of NICD from 2 experiments are indicated in the bottom of the panel. *P < 0.05 (2-tailed Student’s t test) (C) Coexpression of dominant negative human USP35 in bbs4 morphants ameliorates CE defects. Zebrafish embryos were injected with bbs4 morpholino and mRNA encoding either WT or dominant negative human USP35. CE defects can be ameliorated by coexpression of hUSP35 (C450A) but not hUSP35 (WT). ****P < 0.0001 (χ2 analysis). (D) Loss of bbs4 leads to the atrophy and deficit convolution in the proximal tubule. Coexpression of hUSP35 (C450A) but not hUSP35 (WT) can ameliorate the renal development defects. ****P < 0.0001 (χ2 analysis). (E) Represented images of D. Scale bar: 100 μm.