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Functional Characterization of RING-Type E3 Ubiquitin Ligases In Vitro and In Planta
Published on: December 5, 2019
Transcriptional coregulator SNURF (RNF4) possesses ubiquitin E3 ligase activity
M Häkli1, K L Lorick, A M Weissman
1Biomedicum Helsinki, Institute of Biomedicine, University of Helsinki, FIN-00014 Helsinki, Finland.
FEBS Letters
|February 28, 2004
Summary
SNURF/RNF4 acts as an E3 ligase, selectively ubiquitylating itself. This activity, dependent on specific ubiquitin-conjugating enzymes (E2s), is crucial for its transcriptional regulation and growth inhibition functions.
Area of Science:
- Molecular Biology
- Biochemistry
- Cell Biology
Background:
- SNURF/RNF4 is known for transcriptional regulation and growth inhibition.
- Its function is dependent on its RING finger domain.
Purpose of the Study:
- To investigate the E3 ligase activity of SNURF/RNF4.
- To determine the selectivity of SNURF/RNF4 for ubiquitin-conjugating enzymes (E2s).
- To elucidate the relationship between SNURF/RNF4 ubiquitination and its biological functions.
Main Methods:
- In vitro ubiquitination assays using SNURF/RNF4 and various E2 enzymes.
- Site-directed mutagenesis of conserved cysteine residues in the SNURF/RNF4 RING finger.
- Size fractionation of cellular proteins from F9 cells.
Main Results:
- SNURF/RNF4 functions as an E3 ligase, mediating its own ubiquitination in an E2-selective manner.
- SNURF/RNF4 interacts with UbcH5A, UbcH5B, HHR6B, E2-25K, MmUbc7, and UbcH13, but not UbcH3, UbcM4, MmUbc6, or E2-20K.
- Ubiquitination can be mono- or multi-ubiquitination depending on the E2 enzyme.
- Mutating conserved cysteine residues abolished ubiquitination.
- Endogenous SNURF/RNF4 exists in large protein complexes (>500 kDa).
Conclusions:
- SNURF/RNF4 possesses E3 ligase activity, which is closely linked to its transcriptional regulatory functions.
- The E2 selectivity and complex formation are key aspects of SNURF/RNF4's ligase activity.
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