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

• Published in: Proceedings of the National Academy of Sciences - PNAS Vol. 117; no. 49; pp. 31094 - 31104
• Main Authors: Vua, Tri T. M., Mitchell, Dylan C., Gygi, Steven P., Varshavsky, Alexander
• Format: Journal Article
• Language: English
• Published: United States National Academy of Sciences 08-12-2020
• Subjects: Actin; Activating transcription factor 3; Activating Transcription Factor 3 - genetics; Adrenergic receptors; Antigens, Neoplasm - genetics; Biological Sciences; Cell lines; Eye Proteins - genetics; Gene expression; Gene Expression Regulation - genetics; Gene Knockout Techniques; Gene sequencing; Glucocorticoids; HEK293 Cells; Homeobox; Homeodomain Proteins - genetics; Humans; Intermediate Filaments - genetics; Lactic acid; Mass Spectrometry; Mass spectroscopy; Microfilament Proteins - genetics; Monocarboxylic Acid Transporters - genetics; Neoplasm Proteins - genetics; Protein Binding - genetics; Proteins; Proteolysis; Receptors; Receptors (physiology); Receptors, Adrenergic, beta-2 - genetics; Receptors, Glucocorticoid - genetics; Ribonucleic acid; RNA; RNA-Seq; Signal Transduction - genetics; Substrates; Transcription factors; Transcription Factors - genetics; Ubiquitin; Ubiquitin - genetics; Ubiquitin-protein ligase; Ubiquitin-Protein Ligases - genetics; PREP1; degradation; GR; UBR1; transcription
• ISSN: 0027-8424; 1091-6490
• DOI: 10.1073/pnas.2020124117

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.