Welcome to the new version of CaltechAUTHORS. Login is currently restricted to library staff. If you notice any issues, please email coda@library.caltech.edu
Published December 8, 2020 | Supplemental Material + Published
Journal Article Open

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


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.

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.

Attached Files

Published - 31094.full.pdf

Supplemental Material - pnas.2020124117.sapp.pdf


Files (4.9 MB)
Name Size Download all
3.5 MB Preview Download
1.5 MB Preview Download

Additional details

August 22, 2023
December 22, 2023