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Research Article

The Arg/N-degron pathway targets transcription factors and regulates specific genes

View ORCID ProfileTri T. M. Vu, Dylan C. Mitchell, Steven P. Gygi, and Alexander Varshavsky
PNAS December 8, 2020 117 (49) 31094-31104; first published November 23, 2020; https://doi.org/10.1073/pnas.2020124117
Tri T. M. Vu
aDivision of Biology and Biological Engineering, California Institute of Technology, Pasadena, CA 91125;
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  • ORCID record for Tri T. M. Vu
Dylan C. Mitchell
bDepartment of Cell Biology, Harvard Medical School, Boston, MA 02115
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Steven P. Gygi
bDepartment of Cell Biology, Harvard Medical School, Boston, MA 02115
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Alexander Varshavsky
aDivision of Biology and Biological Engineering, California Institute of Technology, Pasadena, CA 91125;
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  • For correspondence: avarsh@caltech.edu
  1. Contributed by Alexander Varshavsky, October 15, 2020 (sent for review September 25, 2020; reviewed by Thomas Arnesen and William P. Tansey)

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Significance

The Arg/N-degron pathway targets proteins for degradation by recognizing their N-terminal or internal degradation signals. In the present study, we compared wild-type human cells to their double-knockout (2-KO) counterparts that lacked the UBR1/UBR2 ubiquitin ligases of the Arg/N-degron pathway. We found that a number of specific genes were either strongly induced or strongly repressed in 2-KO cells. In addition, specific transcription factors, including glucocorticoid receptor, were identified here as physiological substrates of the Arg/N-degron pathway. We discuss the emerged role of this proteolytic system as a regulator of mammalian gene expression.

Abstract

The Arg/N-degron pathway targets proteins for degradation by recognizing their N-terminal or internal degrons. Our previous work produced double-knockout (2-KO) HEK293T human cell lines that lacked the functionally overlapping UBR1 and UBR2 E3 ubiquitin ligases of the Arg/N-degron pathway. Here, we studied these cells in conjunction with RNA-sequencing, mass spectrometry (MS), and split-ubiquitin binding assays. 1) Some mRNAs, such as those encoding lactate transporter MCT2 and β-adrenergic receptor ADRB2, are strongly (∼20-fold) up-regulated in 2-KO cells, whereas other mRNAs, including those encoding MAGEA6 (a regulator of ubiquitin ligases) and LCP1 (an actin-binding protein), are completely repressed in 2-KO cells, in contrast to wild-type cells. 2) Glucocorticoid receptor (GR), an immunity-modulating transcription factor (TF), is up-regulated in 2-KO cells and also physically binds to UBR1, strongly suggesting that GR is a physiological substrate of the Arg/N-degron pathway. 3) PREP1, another TF, was also found to bind to UBR1. 4) MS-based analyses identified ∼160 proteins whose levels were increased or decreased by more than 2-fold in 2-KO cells. For example, the homeodomain TF DACH1 and the neurofilament subunits NF-L (NFEL) and NF-M (NFEM) were expressed in wild-type cells but were virtually absent in 2-KO cells. 5) The disappearance of some proteins in 2-KO cells took place despite up-regulation of their mRNAs, strongly suggesting that the Arg/N-degron pathway can also modulate translation of specific mRNAs. In sum, this multifunctional proteolytic system has emerged as a regulator of mammalian gene expression, in part through conditional targeting of TFs that include ATF3, GR, and PREP1.

  • transcription
  • degradation
  • UBR1
  • GR
  • PREP1

Footnotes

  • ↵1To whom correspondence may be addressed. Email: avarsh{at}caltech.edu.
  • Author contributions: T.T.M.V., D.C.M., S.P.G., and A.V. designed research; T.T.M.V. and D.C.M. performed research; T.T.M.V., D.C.M., S.P.G., and A.V. analyzed data; and T.T.M.V., D.C.M., S.P.G., and A.V. wrote the paper.

  • Reviewers: T.A., University of Bergen; and W.P.T., Vanderbilt University.

  • The authors declare no competing interest.

  • This article contains supporting information online at https://www.pnas.org/lookup/suppl/doi:10.1073/pnas.2020124117/-/DCSupplemental.

Data Availability.

All relevant data are included in the article and supporting information.

Published under the PNAS license.

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The Arg/N-degron pathway targets transcription factors and regulates specific genes
Tri T. M. Vu, Dylan C. Mitchell, Steven P. Gygi, Alexander Varshavsky
Proceedings of the National Academy of Sciences Dec 2020, 117 (49) 31094-31104; DOI: 10.1073/pnas.2020124117

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The Arg/N-degron pathway targets transcription factors and regulates specific genes
Tri T. M. Vu, Dylan C. Mitchell, Steven P. Gygi, Alexander Varshavsky
Proceedings of the National Academy of Sciences Dec 2020, 117 (49) 31094-31104; DOI: 10.1073/pnas.2020124117
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