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Abstract
Centriole duplication is tightly controlled to occur once per cell cycle, and disruption of this synchrony causes centriole amplification, which is frequently observed in many cancers. Our previous work showed that nuclear distribution gene C (NudC)-like protein 2 (NudCL2) localizes to centrosomes; however, little is known about the role of NudCL2 in the regulation of centrosome function. Here, we find that NudCL2 is required for accurate centriole duplication by stabilizing the E3 ligase HECT domain and RCC1-like domain-containing protein 2 (HERC2). Knockout (KO) of NudCL2 using CRISPR/Cas9-based genome editing or depletion of NudCL2 using small interfering RNA causes significant centriole amplification. Overexpression of NudCL2 significantly suppresses hydroxyurea-induced centriole overduplication. Quantitative proteomic analysis reveals that HERC2 is downregulated in NudCL2 KO cells. NudCL2 is shown to interact with and stabilize HERC2. Depletion of HERC2 leads to the similar defects to that in NudCL2-downregulated cells, and ectopic expression of HERC2 effectively rescues the centriole amplification caused by the loss of NudCL2, whereas the defects induced by HERC2 depletion cannot be reversed by exogenous expression of NudCL2. Either loss of NudCL2 or depletion of HERC2 leads to the accumulation of ubiquitin-specific peptidase 33 (USP33), a centrosomal protein that positively regulates centriole duplication. Moreover, knockdown of USP33 reverses centriole amplification in both NudCL2 KO and HERC2-depleted cells. Taken together, our data suggest that NudCL2 plays an important role in maintaining the fidelity of centriole duplication by stabilizing HERC2 to control USP33 protein levels, providing a previously undescribed mechanism restraining centriole amplification.
Highlights
Centrioles are cylindrical, microtubule-based structures that are essential for the formation of centrosomes, cilia, and flagella[1,2]
Subsequent results revealed that nuclear distribution gene C-like protein 2 (NudCL2) was associated with green-fluorescent protein (GFP)-centrin that was stably expressed in HeLa cells (Fig. 1c)
Given that cell cycle arrest may induce centriole amplification[2,11], we determined whether centriole amplification induced by NudCL2 deletion resulted from a change in cell cycle progression in NudCL2 KO cells
Summary
Centrioles are cylindrical, microtubule-based structures that are essential for the formation of centrosomes, cilia, and flagella[1,2]. A pair of centrioles recruits and organizes the pericentriolar material (PCM) to form a mature centrosome, which plays important roles in regulating cell shape, polarity, motility, mitosis, and cytokinesis[1,3]. Nuclear distribution gene C (NudC)-like protein 2 (NudCL2) was cloned and characterized as a new Official journal of the Cell Death Differentiation Association. In the filamentous fungus Aspergillus nidulans, NudC was first identified as an upstream regulator of NudF (a homolog of the human lissencephaly 1 gene product, LIS1) in the control of nuclear movement[7,8]. Our previous study has reported that NudCL2 plays an important role in regulating the LIS1/dynein pathway by enhancing the interaction between LIS1 and heat-shock protein 90 (Hsp90) to stabilize LIS16. NudCL2 has been shown to be associated with centrosomes in human cells[6]; little is known about the potential role of NudCL2 in centrosomes
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