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Simultaneous Disruption of Both Polyubiquitin Genes Affects Proteasome Function and Decreases Cellular Proliferation

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Abstract

The ubiquitin (Ub) proteasome system is important for maintaining protein homeostasis and has various roles in cell signaling, proliferation, and cell cycle regulation. In mammals, Ub is encoded by two monoubiquitin and two polyubiquitin genes. Although reduced levels of Ub due to the disruption of one polyubiquitin gene are known to decrease cell proliferation, the effect of disrupting both polyubiquitin genes remains elusive. Polyubiquitin gene Ubc knockout mice are embryonically lethal and polyubiquitin gene Ubb knockout mice are infertile. Thus, it is difficult to study the effects of double knockouts (DKOs). In the present study, the CRISPR/Cas9 system was used to simultaneously knockout both polyubiquitin genes, UBB and UBC, in HEK293T and HeLa cells. In DKO cells, growth decreased significantly compared to the control cells. We observed reduced proteasome function and reduced levels of free Ub in DKO cells. However, the levels of purified proteasome were not different between control and DKO cells, although the mRNA levels of proteasomal subunits were significantly increased in latter. We propose that the reduction of Ub levels, by disruption of both polyubiquitin genes, resulted in an altered proteasomal status, leading to the reduced proteasome activity, and decreased cellular proliferation.

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Abbreviations

Ub:

Ubiquitin

DKO:

Double knockout

UPS:

Ubiquitin proteasome system

MCM2:

Minichromosome maintenance protein 2

PTM:

Posttranslational modification

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Acknowledgements

This work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government (MSIT) (Nos. 2019R1F1A1061484; 2020R1F1A1070847) to K.-Y.R.

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  1. These authors contributed equally: Chul-Woo Park, Jin-Sil Bae

Authors and Affiliations

  1. Department of Life Science, University of Seoul, Seoul, 02504, Republic of Korea

    Chul-Woo Park, Jin-Sil Bae & Kwon-Yul Ryu

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  1. Chul-Woo Park
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  2. Jin-Sil Bae
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Correspondence to Kwon-Yul Ryu.

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Park, CW., Bae, JS. & Ryu, KY. Simultaneous Disruption of Both Polyubiquitin Genes Affects Proteasome Function and Decreases Cellular Proliferation. Cell Biochem Biophys 78, 321–329 (2020). https://doi.org/10.1007/s12013-020-00933-2

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