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

Vu, Tri T. M. and Mitchell, Dylan C. and Gygi, Steven P. and Varshavsky, Alexander (2020) Arg/N-degron pathway targets transcription factors and regulates specific genes. Proceedings of the National Academy of Sciences of the United States of America, 117 (49). pp. 31094-31104. ISSN 0027-8424. https://resolver.caltech.edu/CaltechAUTHORS:20201124-103211964

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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.


Item Type:Article
Related URLs:
URLURL TypeDescription
https://doi.org/10.1073/pnas.2020124117DOIArticle
https://www.pnas.org/content/suppl/2020/11/19/2020124117.DCSupplementalPublisherSupporting Information
ORCID:
AuthorORCID
Vu, Tri T. M.0000-0002-1098-5646
Gygi, Steven P.0000-0001-7626-0034
Varshavsky, Alexander0000-0002-4011-258X
Additional Information:© 2020 National Academy of Sciences. Published under the PNAS license. Contributed by Alexander Varshavsky, October 15, 2020 (sent for review September 25, 2020; reviewed by Thomas Arnesen and William P. Tansey). PNAS first published November 23, 2020. We are grateful to I. Antoshechkin for RNA-seq analyses. T.T.M.V. and A.V. thank current and former members of the A.V. laboratory for their advice and assistance. D.C.M. and S.P.G. thank J. Paulo for assistance with mass spectrometry experiments. This work was supported by NIH grants 1R01DK039520 and 1R01GM031530 (A.V.) and R01GM067945 (S.P.G.). Data Availability: All relevant data are included in the article and supporting information. 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.
Funders:
Funding AgencyGrant Number
NIH101DK039520
NIH1R01GM031530
NIHR01GM067945
Subject Keywords:transcription; degradation; UBR1; GR; PREP1
Issue or Number:49
Record Number:CaltechAUTHORS:20201124-103211964
Persistent URL:https://resolver.caltech.edu/CaltechAUTHORS:20201124-103211964
Official Citation: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
Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:106809
Collection:CaltechAUTHORS
Deposited By: Tony Diaz
Deposited On:24 Nov 2020 18:51
Last Modified:09 Dec 2020 22:30

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