16 Feb 2023
by Richa

Summary- paper 17: Orphan quality control shapes network dynamics and gene expression

Kevin G. Mark, SriDurgaDevi Kolla, Danielle M. Garshott, Brenda Martínez-González, Christina Xu, David Akopian, Diane L. Haakonsen, Stephanie K., Michael Rapé

Biorxiv, 2022

Questions/gaps addressed:

  • Accurate protein complex formation (common examples of dimerization through Zinc-fingers, BTB domains or leucine zippers) is important for the transcriptional programs that specify cell fate. How are the dynamics of these interactions accomplished?

  • Transcription factors of the BTB family co-translationally form homodimers that are very stable. Transcription factors also contain activation domains that are rich in IDRs that allow recruitment of many proteins and nucleate transcription hubs which stimulate gene expression and controls cell fate decisions. How do cells establish the proper composition and dynamics of intricate transcription hubs?

  • E3 ligase UBR5 was identified to play a role in preserving pluripotency in human embryonic stem cells (hESC). OE of Ubr5 in UBR5 gene in breast/ ovarian cancers s tumor growth and metastasis, and inactivating mutations in UBR5 drive mantle cell lymphoma. How does Ubr5 regulate stem cell identity and act as either a tumor suppressor or an oncogene?

Key methods:

  • Binding partners of endogenously FLAG tagged UBR5 by affinity purification and CompPASS mass spectrometry. Found several subunits of the INO80 complex, DSIF complex, mitotic checkpoint complex (MCC). Confirmed most by Western blotting using antibodies against endogenous proteins.

  • Test if UBR5 induces the turnover of proteins that cooperate with c-MYC, fused interactors to GFP, followed by mCherry after an internal ribosome entry site. The ratio between GFP and mCherry, was measured by fluorescence associated cell sorting (FACS). Monitored effect on stability upon siRNA against UBR5 and found that it degrades many transcriptional regulators that have links to c-MYC.

  • Inhibition of p97 (NMS873), stabilized all targets. Inhibition of proteasome with MG132 also stabilized all targets. Inhibition of lysosomal function with bafilomycin A, had no effect.

  • Tested specificity of UBR5 by comparing upon downregulation of other E3 ligases HUWE1, UBR4, RNF126, CHIP, WWP1, RNF168. See some overlap but UBR5 seems to be specific for transcription factors linked to c-myc.

  • reconstituted the activity of this UBR5 E3 ligase in vitro. Purified UBR% from HeLa cells, and then tested in vitro ubiquitination.

  • Using MCRS1 as a model substrate, tested the linkage type, and found K48 and K63 Ub both were required for UBR5 mediated ubiquitination.

  • Ubiquitylation was strongly enhanced if we bypassed chain initiation by fusing ubiquitin to MCRS1, but blocked if they fused a K48 Ub, suggesting the K63 branches are added to K48 Ub

  • Performed RNAseq in ΔUBR5 cells to identify genes that are upregulated. Found effects on several targets of c-MYC (both up and downregulated) and growth factors important for stem cell pluripotency.

  • To test if UBR5 directly recognizes the candidate c-MYC degrons, synthesized each motif as a TAMRA-labeled peptide and incubated them with UBR5 and UBE2L3. c-MYC contains two carboxy-terminal degrons that are each sufficient to mediate recognition by UBR5.

  • expressed UBR5-targets with dimerization partners and monitored effects on UBR5-dependent degradation. Complex formation stabilized all the cases shown.

Major takeaways:

  • UBR5 is a nuclear HECT E3 ligase. IT synthesizes branched ubiquitin chains, requires the E1 enzyme UBA1 and the Cys-specific E2 UBE2L3.

  • UBR5 targets degrons in the carboxy-terminal domain of c-MYC that become inaccessible upon formation of DNA-bound c-MYC/MAX dimers.